Confocal optical scanning device

ABSTRACT

Confocal optical scanning device designed to allow visualization of an object to be observed ( 1107 ) and comprising: an optical system designed, on the one hand, to focus an illuminating beam (FX) coming from a light source on to at least one illuminating point (FXO) intended to illuminate a point of the object to be observed ( 1107 ) and, on the other hand, to focus a light beam (FE) coming from the illuminated point of the object ( 1107 ) on to a luminous point (FEO) in a first image plane (PI); at least one rotatably mounted mobile mirror ( 1104 ) on which are reflected, on the one hand, the illuminating beam (FX) to allow the scanning of the object to be observed ( 1107 ) along an observed plane and, on the other hand, the light beam (FE) to bring the luminous point (FEO) on to a fixed point on the first image plane; and a first spatial filtering system ( 1203 ) arranged in the first image plane and designed to filter the luminous point (FEO) to obtain a beam to be detected (FD). The optical system and the first spatial filtering system ( 1203 ) are designed to send back the beam to be detected (FD) on to said rotatably mounted mobile mirror ( 1104 ), the optical system is also designed to focus said beam to be detected (FD), on to a point to be detected (FDO) in a second image plane (P 2 ) to obtain, in said second image plane, a movement of said point to be detected proportional to the movement of the illuminated point in the observed plane of the object.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No10/380,373 filed on Jul. 23, 2003 now U.S. Pat. No. 7,209,287 which wasthe National Stage of International Application No PCT/FR01/02890 filedSep. 18, 2001. The entire contents of both these applications isexpressly incorporated herewith by reference thereto.

This invention relates to a confocal optical scanning device used forexample in a fast confocal microscope, and in particular in afluorescence or reflectance confocal microscope designed for real-timeoperation.

The principle of the confocal scanning optical microscope is theillumination of an object to be observed with an illuminating beamfocused on a point of the object, and detecting the light returning fromthis illuminated point of the object. The confocal microscope thus hasan illuminating beam, that may be filtered by a pinhole, and the lightbeam coming from the illuminated object itself goes through a pinholeselecting the light coming from the illuminated point. The light beamfrom the object, and which has been filtered spatially by the pinhole,is then detected.

To generate an image of the object, it is necessary to move theilluminated point of the object in a plane. This scanning can be carriedout in different ways:

-   i)- By moving the observed object in relation to the objective. This    is the simplest method but it is excessively slow. It is described    for example in U.S. Pat. No. 3,013,467.-   ii)- By providing pinholes on a disk which is traversed in one    direction by the illuminating beam and in the other by the light    beam coming from the object. When the disk is rotating rapidly in    the image plane, the pinholes move and the object is scanned. This    is the technique of the Nipkow disk, described for example in U.S.    Pat. Nos. 3,517,980 and 4,927,254. As the disk is pierced with many    holes, this scanning technique is very fast. Detection can take    place directly on a camera, and it is also possible to observe the    image directly with the naked eye. On the other hand, 95% of the    available luminous power is lost, thus limiting the effectiveness of    this technique when samples exhibit low fluorescence.-   iii)- Using galvanometric mirrors to deflect the illuminating beam,    in accordance with a technique described for example in U.S. Pat.    No. 3,518,014. The object is then scanned point by point and a    single pinhole is used. A photomultiplier tube allows the detection    of photons going through this hole. The technique is the one most    used in fluorescence confocal microscopy because there is little    loss of luminous energy. On the other hand, the imaging speed is    limited by the saturation of fluorescence and/or the oscillation    frequency of the galvanometric mirrors. The image cannot be observed    directly and must necessarily be reconstituted by computer based on    data acquired by the photomultiplier tube. The reconstitution of a    horizontal plane or an observed plane of the object requires precise    knowledge of the position of the galvanometric mirrors corresponding    to the signal sampled at each instant on the photodetector. To    operate correctly the system requires very precise control of the    galvanometric mirrors and perfect synchronization between the    movement of the galvanometric mirrors and the sampling of the signal    coming from the photodetector.

The Nipkow disk technique is today the only one making it possible toobtain directly, by optical means, a confocal image that can be recordedon a camera. It however involves a certain number of difficulties. Animportant point is that it is not possible to exchange the Nipkow diskwithout rebalancing the rotary system and, in practice, Nipkow disks arealways fixed, thus generally preventing the optimization of the spatialfiltering characteristics (the width of the pinholes or their density)according to the wavelength used and/or the speed desired. For thisreason, U.S. Pat. No. 6,002,509 proposes a solution for modifying thespatial filtering characteristics by superposing on the same diskseveral zones having different spatial filtering characteristics. Tofilter the beam spatially, that patent uses a transparent disk having anarray of microscopic mirrors which have a function equivalent to thepinholes of an opaque Nipkow disk. Another difficulty has to do with theproblem of stray light which interferes with the useful light, comingfrom the object and filtered by the disk. U.S. Pat. No. 3,517,980 makesit possible to solve this problem at the cost of disk alignmentdifficulties and stability requirements which are difficult to meet.U.S. Pat. No. 4,927,254 only partially solves the problem but preventsany alignment difficulty.

To improve the imaging speed, a Nipkow disk microscope has been designedwith a collecting disk consisting of an array of microlenses, and whichis attached to the disk which has pinholes. Each microlens is placedopposite a pinhole and focuses on this hole the light coming from theilluminating beam. This technique is described for example in U.S. Pat.No. 5,162,941, as well as in U.S. Pat. No. 5,579,157. This techniquemakes it possible to prevent the loss of light power resulting from theuse of a Nipkow disk, while preserving the high imaging speed inherentin Nipkow disk systems.

The technique described in U.S. Pat. No. 5,579,157 raises difficultmechanical design and alignment problems and it is costly to implement.The disk bearing the pinholes and the collector disk must be aligned inrelation to each other with great accuracy. The assembly made up ofthese two disks must rotate rapidly around an axis which must beperfectly fixed, perfectly orthogonal to the plane of the disks, andperfectly orthogonal to the direction of propagation of the incidentlight. In practice, it is extremely difficult to keep the position ofthe axis fixed, and this results in image defects, typically small lightor dark lines superposed on the image. In the above-mentioned systems,part of the illuminating beam is reflected on the plate bearing thepinholes and is then directed to the CCD sensor. As this part of theilluminating beam is very intense in relation to the return light beamcorresponding to the fluorescence, it is difficult to eliminate by meansof dichroic filters, thus resulting in significant attenuation of thebeam.

In addition, the same pinholes are traversed successively by theilluminating beam directed towards the object, and by the light beamcoming from the object. If the diameter of these holes is small, theresulting loss of beam intensity is far greater than that resulting fromsimply reducing the pinhole diameter in a confocal laser scanningmicroscope with galvanometric mirrors. In order for the intensity toremain reasonably high, the pinholes must have a diameter close to thewidth of the Airy disk. This causes a significant reduction inresolution compared with the confocal laser scanning microscope withgalvanometric mirrors, in which resolution can be increased by reducingthe pinhole diameter.

In the above-mentioned systems, the image of a pinhole on the CCDcamera, when this image is moved by a length equal to half the Airy diskwidth, covers quite amply the same image before movement. Consequently,resolution is limited only by the fluorescent emission wavelength, andthe resolution limit is higher than what it is in a confocal laserscanning microscope with galvanometric mirrors, in which advantage istaken of the small excitation wavelength.

Another solution for improving imaging speed is described in U.S. Pat.No. 5,351,152. The system described in that patent comprises a fixedarray of microlenses which split the laser beam into sub-beams which areeach filtered by a pinhole located in an image plane. The objectivefocuses in the object the beams coming from each of these pinholes. Thelight beam re-emitted by the object is then redirected towards a CCDsensor, each pixel of the CCD sensor being the image of a point of theobject on which is focused the beam coming from a corresponding pinhole.Scanning is carried out using the method (i) consisting of moving theobject in relation to the objective, although other methods are notexpressly excluded. The point of the object which is illuminated by oneof the sub-beams and whose image is obtained on a corresponding point ofthe CCD sensor scans a small zone of the object. The image of the objectmust be reconstituted by computer from a series of images obtainedsuccessively on the CCD sensor during the object scanning operation. Thescanning speed of this microscope is thus limited by the reading speedof the CCD sensor, which must be re-read several times to obtain asingle image. A variant of this solution is described in U.S. Pat. No.5,978,095.

In general, the 3D deconvolution of the image, which can substantiallyimprove the resolution, is difficult by means of existing confocalmicroscopes. In scanning instruments with galvanometric mirrors it ismade difficult by sampling errors.

As an example, in FIG. 1 is represented a confocal microscope, accordingto the prior art, using galvanometric mirrors. The illuminating beam FXcoming from a laser 1100 passes through a beam expander composed oflenses 1101 and 1102, is reflected by a splitter 1103, by rotatingmobile mirrors 1104 and 1105, passes through a microscope objective 1106and reaches, after focusing, an illuminating point FXO which illuminatesa point of the object to be observed 1107, this illuminating point FXObeing an isolated point surrounded by a nonilluminated zone. The lightbeam FE, coming from the illuminated point of the object 1107, passesthrough the objective 1106, is reflected by the rotating mobile mirrors1104 and 1105, passes through the splitter 1103 and the lens 1108, andis focused on a luminous point FEO on a spatial filtering system 1220.After passing through the spatial filtering system, the light beam FEIbecomes a beam to be detected FD which is detected by a detector 1221.The spatial filtering system is a pinhole, i.e. it absorbs the lightbeam everywhere except on a hole of small dimensions. In the case of a-Nipkow disk microscope, the Nipkow disk constitutes a spatial filteringdevice selecting the light passing through a set of distinct pinholes.In the case of the microscope described in U.S. Patent Filing 5,239,178,the camera plays both the role of detection system and spatial filteringdevice selecting the light passing through a set of pinholes forming asquare grid array.

FIG. 2 shows a simple ray trace coming from a fixed point 1410 of aplane 1400, along the path of a beam coming from the plane 1400 andreflected by a mobile mirror 1405. After reflection by the mobile mirror1405, the ray trace defines a virtual image plane 1404 conjugate of theplane 1400. The geometrical image of a point 1410 of the plane 1400 is apoint 1411 of the plane 1404. Owing to the rotation of the mobile mirror1410, the geometrical image 1411 of a fixed point 1410 is mobile.

In the case of FIG. 1, a ray trace coming from the point FXO of theobject 1107 and along the path of the beam to be detected defines afirst real image plane P1, in which is placed the spatial filteringsystem 1220, conjugate of the observed plane of the object 1107.

The rotation of the mobile mirror 1104 simultaneously entails:

-   a movement of the image of the plane 1210 in the observed plane of    the object 1107,    -   a movement of the image of the observed plane of the object        1107, in the first image plane, in which is located the spatial        filtering system 1220.

The image FEO in the first image plane P1, of a point FXO made mobile bythe rotation of the mirror 1104, which is itself the image of a fixedpoint 1224, is a fixed point. This usually enables the confocalmicroscope to detect only the light coming from the illuminated pointFXO which scans the object.

Object scanning characteristics depend on the mobile mirrors used. Forexample, if the mirror 1104 is mobile in rotation around an axisorthogonal to the plane of the figure, and the mirror 1105 is fixed,only a line of the observed object can be scanned. If the mirror 1104 ismobile in rotation around two axes orthogonal to each other, or if themirrors 1104 and 1105 are each mobile around an axis, it is possible toscan a plane of the observed object.

Other equivalent configurations can be used although they are not of anyparticular interest and hence are not customarily used. FIG. 3 showssuch a configuration, in which the mobile mirror 1104 in Figure has beenreplaced by a mobile mirror 1230 with several facets, in which aseparator 1232 is placed on the path of the light beam FE coming fromthe object before reflection by a mobile mirror, and in which a mirror1231 is used to send the beam back towards the mobile mirror. Theilluminating beam FX and the light beam FE are reflected by distinctfacets of the mirror, but the optical system is designed so that thebasic properties of the device are conserved. In general, in the presentpatent, the term “mirror” will designate a mirror with one or morefacets, the facets being integral with each other but may be separatedby nonreflecting zones, and the mirror may have very diverse forms.

In the device of FIG. 1, a signal reaching the sensor 1203 is recordedand an image of the observed plane is reconstituted by a computer. Toreconstitute the image, it is necessary to have access, for each sampledvalue of the signal coming from the sensor 1203, to the correspondingposition of the mobile mirrors. This device is thus extremely sensitiveto any error in the positioning of the galvanometric mirrors. Moreover,it does not allow direct observation of the image by means of aneyepiece.

It is an object of the present invention to overcome the technicalproblems mentioned above, by providing a confocal optical scanningdevice allowing the recording of a signal selected by the spatialfiltering system, which permits a reconstitution of the observed objectthat will not be excessively sensitive to galvanometric mirrorpositioning errors. In particular, it is an object of the presentinvention to provide a confocal optical scanning device having, like theNipkow disk systems, the ability to generate an image that can berecorded for example on a matrix CCD sensor using a purely opticalmethod, but in which, unlike the Nipkow disk, the spatial filteringcharacteristics can be easily modified in order to always conserveoptimum image quality.

For this purpose, it is the object of the invention to provide aconfocal optical scanning device including:

a optical system designed, on the one hand, to focus an illuminatingbeam coming from the light source on to at least one illuminating pointisolated and surrounded by a nonilluminated zone, said at-least-oneilluminating point being designed to illuminate a point of the object tobe observed and, on the other hand, to focus a light beam coming fromthe illuminated point of the object on to a luminous point in a firstimage plane;

at least one mirror mounted movably in rotation and on which arereflected, on the one hand, the illuminating beam to allow saidat-least-one illuminating point to scan the object to be observed alongan observed plane and, on the other hand, the light beam coming from theilluminated point of the object to bring the luminous point on to afixed point on the first image plane;

a first spatial filtering system arranged in the first image plane anddesigned to obtain a beam to be detected coming from the filteredluminous point;

wherein the optical system and the spatial filtering system are designedto send the beam to be detected on to said mirror movable in rotation,and wherein the optical system is also designed to focus said beam to bedetected, reflected off the mobile mirror, on to a point to be detectedin a second image plane in order to obtain, in said second image plane,a movement of said point to be detected proportional to the movement ofthe illuminated point in the observed plane of the object.

Thanks to these arrangements, the reflection of the beam to be detectedby the mobile mirror allows the image of the first spatial filteringsystem to move in the second image plane. As the mobile mirror on whichthe reflection of the beam to be detected takes place is the same asthat on which the reflection of the light beam and the reflection of theilluminating beam take place, the movement of the point to be detectedin the second image plane is proportional to the movement of theilluminated point in the observed plane. For example, in the case inwhich the spatial filtering system is a single pinhole as in FIG. 1, itis possible to place a linear or matrix CCD sensor in the second imageplane, which must then be real. To each position of the image of thepinhole on the sensor there then corresponds a position of the observedpoint in the observed plane. On the image formed on the sensor, eachpoint corresponds to a perfectly determined point of the observedobject, the position of which is not affected by any errors in thepositioning of the mobile mirror. The data recorded on the sensor thusmake it possible to reconstitute faithfully the image of a scanned linein the object (if the rotation of the mirror takes place around a singleaxis) or a scanned plane in the object (if the rotation of the mobilemirror takes place, for example, around two axes orthogonal to eachother), even if the mobile mirror positioning control is not perfect.

The second image plane may be real or virtual. However, the aim is tofocus the beam to be detected in a real image plane which can beincluded in the scanning device or outside this device. This real imageplane may, for example, be on the retina of the eye of an observer or onthe sensor of a camera.

According to another characteristic of the invention, the optical systemincludes means for splitting the illuminating beam into a plurality ofilluminating sub-beams, and the optical system is designed, on the onehand, to focus the plurality of illuminating sub-beams into a pluralityof corresponding illuminating points on the observed plane of the objectand, on the other hand, to focus the plurality of light sub-beams comingfrom the plurality of illuminated points of the object into a pluralityof luminous points on the first spatial filtering system, and the firstspatial filtering system is designed to filter individually eachluminous point coming from each illuminated point of the object in orderto obtain a plurality of corresponding sub-beams to be detected.

Thus, the system according to the invention makes it possible to achievemultipoint confocal imaging having all the advantages, in terms ofspeed, of known solutions such as the Nipkow disk microscope.

However, in this case where a multipoint technique is used, the imagepoints of each illuminated point of the observed plane movesimultaneously in the second image plane, and a point of the secondimage plane may be illuminated successively by the light coming from twodifferent points of the observed object and having passed through twodistinct points of the first spatial filtering system. The lightintensity recorded at a point of the second image plane then correspondsto the superposition of signals coming from two distinct points of theobject, and consequently does not allow optimum reconstitution of theobserved object.

According to yet another characteristic of the invention, this problemis solved by adapting the optical system so that the geometrical imageof a fixed geometrical point of the object, in the second image plane,is independent of the position of said at-least-one mirror mountedmovably around its axes of rotation. In fact, in this case, a point ofthe second image plane is illuminated only by the light coming from itsfixed geometrical image in the object, thus allowing the formation of agood-quality image. Preferably, the system may be adapted so that thisgeometrical image is fixed for arbitrary rotary movements of the mirroraround three noncoplanar axes, including the case in which the mirrorhas only one axis of rotation. This prevents vibrations andinstabilities of the mirror from resulting in movements of the image ofa point in the illuminated plane of the object, and hence in samplingerrors.

According to a characteristic of the invention, the scanning devicecomprises means for modifying the spatial filtering characteristics ofthe first spatial filtering system. This is facilitated by the fact thatthe first spatial filtering system is static during image acquisition,unlike a Nipkow disk. By spatial filtering characteristics is meant, forexample, the diameter of each pinhole or of each microscopic mirrorconstituting the spatial filtering device, or the distribution of theseelements on the spatial filtering device. For example, the first spatialfiltering system may be designed with a spatial light modulatorconsisting of a matrix-type or ferroelectric liquid crystal-type device.In this case, the electrical connection of this device is facilitated bythe fact that it is static. However, it is generally more effective touse mechanical means to modify the spatial filtering characteristics.According to a characteristic of the invention, the first spatialfiltering system comprises at least one mobile or movable element tomodify the spatial filtering characteristics of the first spatialfiltering system. For example, the first spatial filtering system(130;606) may be mounted movably to be replaced, manually or by means ofa motor-driven system, by another first spatial filtering system. Thefirst spatial filtering system may also comprise several distinct zoneswhose filtering characteristics differ from each other, and be mountedmovably to enable either one or the other of these zones to be placed onthe path of the light beam. It may also be made up of a first absorbingand/or reflecting plate designed with a plurality of pinholes, and asecond plate comprising absorbing and/or reflecting parts andtransparent parts, these two planes being placed against each other andmobile in relation to each other so that the absorbing and/or reflectingparts of the second plate obstruct part of the pinholes of the firstplate, and so that the relative movement of the two plates allows themodification of the pinholes left free. Whatever the method used, thedevice according to the invention makes it possible to modify thespatial filtering characteristics, thus allowing the optimization ofthese characteristics according to the wavelength used and/or theimaging speed or imaging quality desired. The movement of the mobile ormovable element of the spatial filtering system requires, in the generalcase, good mechanical accuracy and/or a suitable alignment procedure:if, for example, the first spatial filtering system comprises pinholesabout 30 microns wide, it is desirable that the positioning accuracy bebetter than 5 microns. On the other hand, as the first spatial filteringsystem is static during the operation of the microscope, it does notbecome misaligned and does not raise any balancing problems, thussolving the problem encountered in Nipkow disk systems; in the presentscanning device, it is possible to modify the spatial filteringcharacteristics by a simple mechanical means. In the case, for example,in which the spatial filtering device is mounted movably and isexchangeable manually, there is no limit to the number of differentdevices that may be exchanged, and it is thus possible to optimize thefiltering characteristics. If the spatial filtering device compriseszones and is mobile, the number of zones may be far greater than thatwhich is utilizable on a Nipkow disk of the type described in U.S. Pat.No. 6,002,509. In fact, this number of zones is proportional to thetotal surface area of the device, whereas on a Nipkow disk it isproportional to the radius of the disk, i.e. to the square root of thesurface area.

According to another characteristic of the invention, the means forsplitting the illuminating beam into a plurality of illuminatingsub-beams consist of the first spatial filtering system. Thisarrangement generally allows the system to be simplified. In particular,when the first spatial filtering system is exchangeable, thisarrangement allows a considerable reduction in alignment stresses andimproved mechanical accuracy. In fact, in this case, the first spatialfiltering system determines both the position of the illuminated pointsin the sample or the object and the position of the filtered points toobtain the beam to be detected. When the spatial filtering system isexchanged, the position of the filtered luminous points correspondsautomatically to the position of the illuminated points, withoutrequiring any high-precision adjustments.

If the first spatial filtering system also constitutes the means forsplitting the illuminating beam into a plurality of illuminatingsub-beams, and if this spatial filtering system is made up of a platepierced with pinholes, the optical system required to bring the beam tobe detected towards the mobile mirror is relatively complex. Thisoptical system may be considerably simplified, according to anotheradvantageous characteristic of the invention, when the spatial filteringdevice is composed of a plate designed with a plurality of microscopicmirrors, and allowing the sending of the beam to be detected in adirection close to the direction of the incident beam. This solutionmoreover has the advantage, if the plate is transparent, of reducing theamount of light coming from the reflection of the illuminating beam onthe spatial filtering system and which is superposed on the beam to bedetected. In this case, and according to another characteristic of theinvention, the optical system is preferably designed so that only oneface of the rotatably mobile mirror reflects the plurality ofilluminating sub-beams coming from the first spatial filtering system tothe object to be observed, the plurality of the light sub-beams to thefirst spatial filtering system and the plurality of the sub-beams to bedetected to the second image plane. This adaptation may be obtained in aparticularly simple manner, according to another feature of theinvention, by means of a single lens traversed by the plurality ofilluminating sub-beams directed from the first spatial filtering systemto the movably mounted mirror by the plurality of light sub-beamsdirected from the mobile mirror to the first spatial filtering system,and by the plurality of sub-beams to be detected directed from the firstspatial filtering system to said movably mounted mirror. By “lens” ismeant here either, as in the entire text of the patent, a single lens ora compound lens, for example an achromat or a set of several achromatsseparated by air.

It is another object of the invention to allow the elimination of thelight coming from the reflection of the illuminating beam on the spatialfiltering system and which is superposed on the beam to be detected, andthis without the difficulties related to the use of a Nipkow disk. Forexample, in a Nipkow disk-type microscope, the elimination of the lightcoming from the reflection of the illuminating beam on the spatialfiltering system requires the use of two distinct parts of the disktraversed respectively by the illuminating beam and the light beamreflected by the sample, thus causing considerable adjustment andstability problems in the Nipkow disk.

According to one characteristic of the invention allowing the solutionof the above problem, while using in its totality the opening of theobjective, for the illumination beam as well as for the light beamcoming from the object, the optical system also comprises a splitter toseparate the plurality of illumination sub-beams directed to the objectto be observed, from the light beam coming from the object to beobserved to the first spatial filtering system, so that the firstspatial filtering system is not reached by the illumination beam. As thefirst spatial filtering system is not reached by the illumination beam,the stray light is eliminated. However, owing to the fact that the firstspatial filtering system is fixed, this result is obtained without theinstability problems related to the Nipkow disk. To completely avoidstray light reflection on the first spatial filtering system, the firstspatial filtering system is preferably made up of an absorbing and/orreflecting plate bearing pinholes through which light passes in a singledirection. To completely avoid stray light reflections on the means forsplitting the illuminating beam into sub-beams, these illuminating beamsplitting means may preferably be, in the case of illumination innoncoherent light, a plate provided with a plurality of pinholes throughwhich the illuminating beam passes in only one direction. In the case ofillumination with coherent light, and so as not to reduce the usefulintensity of the beam, it is preferable to use a support having aplurality of microlenses. In both cases, operation during transmissionand the use of a splitter make it possible to avoid stray-light relatedproblems.

According to another characteristic of the invention, the means forsplitting the illuminating beam into a plurality of sub-beams include asupport with a plurality of microlenses, and in which the first spatialfiltering system comprises a plurality of pinholes through which theplurality of illuminating sub-beams pass. In this case, and according toa characteristic of the invention, the support comprising a plurality ofmicrolenses, and the first spatial filtering system, are integral witheach other and constitute a splitting and filtering system mountedmovably so as to be replaced by another splitting and filtering system.The advantage of this solution is that, even in the case in which asplitter is used, it is not necessary to integrate this splitter in theremovable splitting and filtering system.

When only one reflection face of a mirror is used, it is necessary, inorder to avoid light intensity losses by polarizing devices allowing theseparation of the different beams, to increase the size of the movablymounted mirror so as to separate spatially some of the beams. Increasingthe size of the mirror results in a decrease of its resonance frequency,which may be troublesome for obtaining clean scanning. In addition, whenthe first spatial filtering system operates under reflection, theoptical system required for the use of a single mirror face is complex.To optimize the resonance frequency of the movably mounted mirror, andin order to simplify the optical system, the movably mounted mirror maybe a plane mirror comprising a first reflection face and a secondreflection face, the first reflection face being designed to deflect theilluminating beam coming from the light source and the light beamemitted by the illuminated point of the object, and the secondreflection face being designed to deflect the beam to be detected sentback by the first spatial filtering system and the optical system.

According to another characteristic of the invention, the means forsplitting the illuminating beam into a plurality of illuminatingsub-beams are designed so that the plurality of illuminating pointsforms a two-dimensional periodic array. Preferably, this array may be ahexagonal grid. Thanks to these arrangements, the device according tothe invention allows light intensity losses to be minimized and thepossibility of obtaining constant imaging characteristics independent ofthe considered point.

In this case, and in order to obtain constant imaging characteristics,it is possible to control said at-least-one mobile mirror so that eachilluminated point completes a two-dimensional scanning of the object.However, this solution calls for efficient mobile mirrors with a highresonance frequency, and a more economical solution is for each point toscan a single line. If this line is curved, for example if each pointscans a circle, the obtained image tends to exhibit illuminationirregularities in certain directions. According to one version of theinvention, a good-quality image benefiting from homogeneous illuminationis obtained by controlling said at-least-one mirror mounted movably inrotation so as to move each of said illuminated points along a straightline not parallel to the direction of the director vectors of theperiodic array. This solution makes it possible in particular to use asingle mirror mounted movably in rotation.

When said at-least-one mobile mirror rotates around its axis or axes,each illuminated point moves in the object, and the entire illuminatedzone moves consequently in the same manner, and all the pointsilluminated during the movement of this zone define an extendedilluminated zone. The points of the object located at the center of theextended illuminated zone are illuminated more often than those locatedon the periphery of this zone, and the illumination is thus nothomogeneous in the extended illuminated zone, thus making it difficultto obtain a good-quality image of a fixed observed zone. According to acharacteristic of the invention, the illuminated zone can be made fixedby means of a diaphragm placed in an image plane through which passesthe illuminating beam coming from said mobile mirror and directed to theobserved object. This diaphragm makes it possible to limit theilluminated zone to the central part of the extended illuminated zonewhich would be obtained in the absence of a diaphragm. However, as partof the illuminating beam is stopped by the diaphragm, this solutionreduces the useful intensity of the illuminating beam.

According to another characteristic of the invention providing asolution to this problem, the device comprises means for moving a zoneilluminated by the illuminating beam on to the means for splitting theilluminating beam into sub-beams. The means for splitting theilluminating beam into sub-beams are, for example, a plurality ofmicrolenses placed in the splitting plane, a transparent and/orreflecting plate designed with a plurality of pinholes and placed in thesplitting plane, or a transparent plate designed with a plurality ofmicroscopic mirrors and placed in the splitting plane. The movement ofthe illuminated zone in the splitting plane allows the modification ofthe illuminated zone in the object by modifying the sub-beams making upthe plurality of sub-beams, but without modifying the characteristics ofthe individual movement of each illuminated point in the object.According to a characteristic of the invention, the means for moving thezone illuminated by the illuminating beam on to the means for splittingthis illuminating beam into sub-are also designed so that theilluminated zone in the observed object remains fixed when saidat-least-one mirror mounted movably in rotation turns. This makes itpossible to conserve a fixed illuminated zone without the loss ofintensity related to the use of a diaphragm. The means for moving theilluminated zone in the splitting plane can, for example, comprise anacoustical-optical beam deflection device or a mobile mirror independentof the one used for moving the illuminated points in the object. In thiscase, the independent mobile mirror or the acoustical-optical devicemust be synchronized with said at-least-one movably mounted mirror.According to a characteristic of the invention, the means for moving thezone illuminated by the illuminating beam in the splitting planecomprise at-least-one mirror mounted movably in rotation, and theoptical system is designed so that the illuminating beam is reflectedbefore reaching the splitting plane by said at-least-one mirror mountedmovably in rotation. As the reflection is carried out on said mobilemirror, it is not necessary to perform any synchronization. Thissolution is, moreover, generally less expensive. However, if the firstspatial filtering device is a plurality of pinholes through which thelight beam passes in a single direction, this solution calls for the useof several additional splitters, and it is technically preferable to usean independent mobile mirror to move the illuminated zone in thesplitting plane. Any synchronization defects in this mirror affect onlythe edges of the illuminated zone and can be eliminated by means of adiaphragm placed in an image plane through which passes the illuminatingbeam coming from said mobile mirror and directed to the observed object.

When the mobile mirrors rotate, the movement speed of the illuminatingpoints in the observed plane varies. These speed variations generatelocalized over-illumination. For example, when the illuminating pointmoves over a line segment that it traverses alternately in one directionand the other, its speed is cancelled at the two ends of the segment,and the point of the observed object which is illuminated when theilluminating point reaches one end of the segment is thusover-illuminated, as a time average, in relation to neighboring points.If the illuminating point crosses successively several distinct linesegments, the same problem exists. In general, any speed variation inthe illuminating point generates illumination nonhomogeneities. Whensaid mobile mirror rotates, the position of an illuminating point in theobject is the mobile geometrical image of a corresponding fixed point onthe first spatial filtering device. When an illuminating point isextinguished (obstructed) by different means, it no longer existsphysically, but the movement it would have if it had not beenextinguished may be characterized by the movement of the geometricalimage of a fixed geometrical point of the spatial filtering device. Tosolve the problem of localized over-illuminations, one will be led toextinguish, by various means, the illuminating beam when the movementspeed of the geometrical image of a fixed geometrical point of thespatial filtering device reaches its minimum value (i.e. when themovement speed of the illuminating points, if they have not yet beenextinguished, reaches its minimum value). According to a characteristicof the invention, the scanning device accordingly comprises means foreliminating the illuminating beam, or the plurality of illuminatingsub-beams, before it reaches the observed object, when the movementspeed of the geometrical image, in the observed object, of a fixedgeometrical point of the first image plane, reaches its minimum value.

According to a characteristic of the invention, the means foreliminating the illuminating beam or the plurality of illuminatingsub-beams can consist of a shutter allowing the extinguishing of theilluminating beam when the movement speed of the illuminating points isat its minimum. However, the extinguishing of the beam must take placeat a very precise moment so that the trajectory of each illuminatingpoint ends at a perfectly defined point, otherwise illuminatingnonhomogeneities are also generated. This synchronization is difficult.

According to yet another characteristic of the invention aimed atsolving the problem of illuminating nonhomogeneities by limiting thesynchronization problems, the means for eliminating the illuminatingbeam or plurality of illuminating sub-beams consist of a limitationdevice designed to be reached by the illuminating beam or plurality ofilluminating sub-beams, said limitation device having means foreliminating part of the illuminating beam or plurality of illuminatingsub-beams not reaching a selection surface, and the limitation device,the optical system, and the movement of the rotatably mounted mobilemirror are designed so that the zone illuminated by the illuminatingbeam on the limitation device is outside the selection surface when themovement speed of the geometrical image, in the observed object, of afixed geometrical point of the first image plane, reaches its minimumvalue. Under these conditions, the illuminating beam is stopped when themovement speed of the illuminating points would normally be at itsminimum value, and the over-illumination of the corresponding points ofthe observed object is prevented. When this solution is applied to asystem in which each illuminating point moves along a straight line, italso enables each illuminating point to traverse entirely the observedzone, so that the beginning and end of its trajectory are defined by thelimits of the observed zone without precise synchronization beingnecessary.

The characteristics of the limitation device differ depending on whetheror not the scanning device comprises means for moving the illuminatedzone on the means for splitting the illuminating beam into a pluralityof sub-beams. In general, the limitation device must be placed in a partof the system in which the zone illuminated by the illuminating beammoves. According to another characteristic of the invention, suited inparticular to the case in which the scanning device comprises means formoving the illuminated zone on means for splitting the illuminating beaminto a plurality of sub-beams, the limitation device is arranged to bereached by the illuminating beam or the plurality of illuminatingsub-beams before the illuminating beam or the plurality of illuminatingsub-beams is reflected, in the direction of the observed object andcoming from means for splitting the illuminating beam into a pluralityof sub-beams, by said rotatably mounted mobile mirror. In this case, thelimitation device may be placed in a plane conjugate to the observedplane so that the observed part of the observed plane is delimited bysharp contours. It may be replaced in the plane in which are located themeans for splitting the illuminating beam into a plurality of sub-beams,which in the case in which microlenses are used is not conjugate to theobserved plane, which in this particular case does not degrade thesharpness of the image's edges. The limitation device can, for example,be a diaphragm in which the limitation surface is the aperture of thisdiaphragm. If the means for splitting the illuminating beam into aplurality of illuminating sub-beams are made up of an absorbing and/orreflecting plate provided with a plurality of pinholes, the limitationdevice can be made up of a part of this plate which does not havepinholes, thus obviating the introduction of an image plan or anadditional diaphragm. If the means for splitting the illuminating beaminto a plurality of illuminating sub-beams consist of a transparentplate provided with a plurality of microscopic mirrors, the limitationdevice can be made up of a part of this plate which does not havemicroscopic mirrors, thus also obviating the introduction of adiaphragm. If the means for splitting the illuminating beam consist of aplurality of microlenses placed in a splitting plane, the limitationdevice may be a diaphragm placed in the same plane or in a focusingplane of the sub-beams coming from the microlenses.

According to a characteristic of the invention, suited in particular tothe case in which the scanning device does not comprise means for movingthe illuminated zone on means for splitting the illuminating beam into aplurality of sub-beams, the limitation device is placed near a planeconjugate to the observed plane and traversed by the illuminating beamafter it has been reflected, coming from means for splitting the beaminto sub-beams and in the direction of the observed object, by therotatably mounted mobile mirror. The limitation device can then be adiaphragm and the selection surface is then the aperture of thisdiaphragm. As previously, in the case in which each illuminating pointmoves along a straight line, each illuminating point crosses entirelythe observed zone. However, this solution may result in a significantloss of luminous intensity.

The scanning device may be considerably simplified when the firstspatial filtering system comprises a nonabsorbing plate provided with atleast one reflecting microscopic mirror, and the rotatably mountedmobile mirror comprises a reflection face which reflects theilluminating beam directed to the object to be observed, the light beamcoming from the object to be observed and directed to the first spatialfiltering system, and the beam to be detected coming from the spatialfiltering system. This solution limits considerably the number of systemcomponents. However, it does not allow the total elimination ofinterference with stray light.

When a single reflection face of a mirror is used, it is necessary, inorder to avoid luminous intensity losses by polarizing devices enablingthe splitting of the different beams, to increase the size of the mobilemirror so as to separate spatially some of the beams. Increasing thesize of the mirror results in a decrease in its resonance frequency,which may be detrimental to proper scanning. In addition, when the firstspatial filtering system works under reflection, the optical systemrequired to use a single face of a mirror is complex. To optimize theresonance frequency of the mobile mirror, and to simplify the opticalsystem, the mobile mirror may be a plane mirror comprising a firstreflection face and a second reflection face, the first reflection facebeing designed to deflect the illuminating beam coming from the lightsource and the light beam emitted by the illuminated point of theobject, and the second reflection face being designed to deflect thebeam to be detected sent by the first spatial filtering system and theoptical system. This configuration is suited particularly to the use ofa first spatial filtering system and comprises an absorbing and/orreflecting plate provided with at least one pinhole traversed in asingle direction by the light beam. This solution can prevent straylight problems, and the use of two opposite faces of a mirror obviatesthe need to re-superpose the filtered beam rid of stray light on theilluminating beam.

Other characteristics and advantages of the invention will appear in thedescription which follows of several of its embodiments, given by way ofnonlimitative examples, in connection with the appended drawings.

In the drawings:

FIGS. 1 and 3 represent prior-art confocal optical scanning devices;

FIG. 2 represents an example of a ray trace coming from a fixed point;

FIG. 4 represents an embodiment using point-by-point scanning and amicroscopic mirror. This embodiment, represented in FIG. 4, is the bestfor an immediate understanding of the operating principle;

FIG. 5 represents an embodiment using point-by-point scanning and apinhole;

FIG. 6 represents an embodiment using multipoint laser illumination anda pinhole array;

FIG. 7 represents a pinhole array used in various embodiments;

FIG. 8 represents a side view of a set of mirrors also represented inFIG. 6;

FIG. 9 represents a lens array used to illuminate the sample;

FIG. 10 represents a set of illuminated points in the observed object,and the trajectory of these points;

FIGS. 11 to 13 illustrate the command applied to a beam attenuatoraccording to the position and movement speed of the image of a pinholeon a camera;

FIG. 14 represents an embodiment of the confocal optical scanning devicein accordance with the invention and in which use is made of multipointlaser illumination and a pinhole array;

FIG. 15 represents a lens array and a pinhole array capable of beingused to filer the light beam coming from the object;

FIG. 16 represents an embodiment using multipoint incoherent lightingand two pinhole arrays;

FIG. 17 represents a cube incorporating two pinhole arrays and adichroic mirror;

FIG. 18 represents an embodiment using multipoint incoherent lightingand a single pinhole array;

FIG. 19 represents the illuminated points in the object, and thetrajectory of one of these points, a single galvanometric mirror beingused;

FIG. 20 represents an embodiment using incoherent lighting a microscopicmirror array, and in which the illuminating beam undergoes a firstdeflection before reaching the microscopic mirror array forming thefirst spatial filtering system;

FIG. 21 represents a microscopic mirror array used in the embodiment ofFIG. 20;

FIG. 22 represents an improved version of the embodiment of FIG. 20, inwhich a nonpolarized beam can be used with no loss of luminous energy;

FIG. 23 represents the movement of an illuminated zone on a transparentplate provided with an array of microscopic mirrors and used as a meansof splitting the illuminating beam into sub

beams, for example in the embodiment of FIG. 22;

FIG. 24 shows the trajectory of an illuminating point in the observedzone of the object;

FIG. 25 represents part of an array of reflecting points with hexagonalgrid, and indicates the position of this array in relation to thedirection of movement of the illuminated zone on the array;

FIG. 26 represents a version with two galvanometric mirrors of theembodiment of FIG. 22;

FIG. 27 represents an embodiment with multipoint laser illumination, inwhich the illuminating beam undergoes a first deflection before reachingthe microlens array;

FIG. 28 shows a simplified embodiment with laser illumination;

FIG. 29 shows an embodiment similar to that of FIG. 28, but with whitelight illumination;

FIG. 30 represents the movement of an illuminated zone on a diaphragm,used for example in the embodiment of FIG. 29;

FIGS. 31 and 32 represent two plates provided with pinholes, both ofthese plates placed one against the other forming a modifiable spatialfiltering device;

FIG. 33 shows a plate equipped with a plurality of pinhole arrays andmobile in one direction, allowing a modification of spatial filteringcharacteristics;

FIG. 34 shows a plate provided with a plurality of pinhole arrays andmobile in two directions, allowing a modification of spatial filteringcharacteristics;

FIG. 35 shows a set of cubes each including a splitter and two spatialfiltering devices, the assembly being mobile in one direction to be ableto change the cube used in the device.

SIMPLE SINGLE-POINT EMBODIMENT

This simple embodiment uses single-point laser illumination in which thespatial filtering system is a microscopic mirror, and in which a singleface of the mobile mirrors is used. Owing to its simplicity, thisembodiment lends itself well to an intuitive understanding of theoperating principle.

FIG. 4 shows a confocal laser scanning fluorescence microscope accordingto this embodiment of the invention. In the figure is indicated in thinlines a beam passing through a point of the object, and in arrows thedirections of the different beams, namely the illuminating beam FX, thelight beam FE and the beam to be detected FD.

An illuminating or excitation beam FX coming from a laser 1100 passesthrough a beam expander or collimator formed by lenses 1101, 1102 thenreflected by the dichroic mirror 1103 which reflects the wavelength ofthe illuminating beam FX of the laser 1100 and allows the passage of thewavelength of the light beam FE retransmitted by fluorescence. Theilluminating beam FX is then reflected on the rotatably mobilegalvanometric mirror 1104 rotating around an axis located in the planeof the figure and in the plane of the mirror 1104, then on the rotatablymobile galvanometric mirror 1105 rotating around an axis orthogonal tothe plane of the figure. It then passes through the polarizing beamsplitter 1204 and then the microscope objective 1106. It is focused bythe objective at an illumination point FXO which illuminates a point ofthe object formed by a fluorescent sample 1107. The light beam FEretransmitted by fluorescence from the illuminated point is collected bythe objective 1106, passes through the polarizing beam splitter 1204, isreflected successively by the two galvanometric mirrors 1105 and 1104,passes through the dichroic mirror 1103, and is focused by the lens 1108at a luminous point FEO. This luminous point FEO is located in a firstimage plane P1 on which is also provided a microscopic mirror 1203located on the rear face of a quarter-wave plate 1202. The microscopicmirror 1203 comprises, in this embodiment, a first spatial filteringsystem for filtering the luminous point. The part of the luminous pointFEO that passes on the side of the microscopic mirror then reaches anabsorbing cavity. The microscopic mirror 1203 can be obtained by opticallithography, for example.

The part of the luminous point FEO or of the beam FE which is reflectedby the point 1203 will hereinafter be called the beam to be detected FD.This beam to be detected FD passes again through the quarter wave plate1202, the lens 1108, the dichroic mirror 1103, is reflected by thegalvanometric mirrors 1104, 1105, is reflected by the polarizing beamsplitter 1204, then focused by the lens 1205 at a point to be detectedFDO in a second image plane P2 on which is provided a CCD sensor 1206fixed on a camera 1207.

Laser polarization 1100 is chosen so that the beam FX coming from thislaser passes through the beam splitter 1204. The quarter-wave plate 1202is a quarter wavelength for the light beam FE retransmitted byfluorescence. Its functions is to rotate by 90 degrees the polarizationdirection, so that only the beam to be detected FD reflected by themicroscopic mirror is then reflected by the polarizing beam splitter1204, to the exclusion of the wave coming from spurious reflections onthe lens 1108.

Under these conditions, the confocal image of the object is formeddirectly on the CCD sensor 1206 when the object is scanned by means ofgalvanometric mirrors. Imperfect control of the galvanometric mirrorsmay result in the worst case in dark zones on the image, but in no casein a displacement of the illuminated points of the object or anygeometrical inaccuracy. The scanning of the object must be carried outduring the sensor integration time. The images can then be transferredfrom the CCD sensor to a sampler or a computer.

The sensor can also be replaced by an eyepiece, possibly a binocularassembly, allowing direct observation of the image formed in the secondimage plane P2 in which is located the CCD 1206 in the diagram. In thiscase, scanning must take place sufficiently fast so as not to beperceptible to the eye.

This embodiment may be suited to a multibeam system, for example of thetype represented in FIG. 20. Its main advantage is that it does notrequire any precise adjustment except for the matching of the laser'sfocusing point with the microscopic mirror. Its major drawback is theloss of luminous intensity resulting from the use of semi-transparent orpolarizing mirrors to allow the use of a single side of the mirrors forall the optical paths.

The present embodiment allows particularly easy understanding of thedevice's operating principle. As in the confocal laser scanningmicroscope represented in FIG. 1, the movement of the galvanometricmirrors results in the scanning of the object 1107 by the illuminatingpoint FXO resulting from the focusing of the illuminating beam FX in theobserved plane of the observed object. The light beam FE emitted byfluorescence then reaches, is fixed and is on the microscopic mirror1203. In the confocal scanning microscope represented in FIG. 1, thismicroscopic mirror is replaced by a pinhole behind which the signal isdetected by means of a photomultiplier. In the present case, the lightbeam FE is reflected by the microscopic mirror 1203, giving the beam tobe detected FD. The beam to be detected FD follows exactly the reversepath of the light beam FE and, in the absence of the beam splitter 1204,would come back exactly to the focusing point FXO of the illuminatingbeam FX in the sample 1107. By introducing the beam splitter 1204, thisbeam is deviated so that it reaches a point FDO of the CCD 1206 locatedin the plane P2. This point FDO of the CCD 1206 is reached only when thecorresponding point FXO of the sample 1107 is illuminated by the beamFX. Each point of the CCD 1206 thus corresponds to a unique point of thesample 1107 and, when the sample 1107 is scanned by means ofgalvanometric mirrors, an image of this sample is formed on the CCD1206. More precisely, in the present case, the geometrical image in theplane P2 of a fixed point of the observed plane of the observed objectis fixed.

Single-point Embodiment Using Opposite Faces of Galvanometric Mirrors

This embodiment is represented in FIG. 5. It is a system usingsingle-point laser illumination, but two faces of the galvanometricmirrors and a pinhole are used. These characteristics prevent most ofthe spurious reflections.

The illuminating beam FX coming from the laser 1300 passes through thebeam expander or collimator made up of lenses 1301, 1302 and is thenreflected by the dichroic mirror 1303. It is then reflected by therotatably mobile galvanometric mirror 1304 rotating around an axislocated at the intersection of the plane of the figure and the plane ofthe mirror 1304, then by the fixed mirror 1305 and by the rotatablymobile galvanometric mirror 1306 rotating around an axis orthogonal tothe plane of the figure. It then passes through the objective 1307forming an image of the sample at infinity, and is focused at anilluminating point FXO which illuminates a point of the observed sample1308. The light beam FE retransmitted by fluorescence from this pointpasses through the objective 1307 in the opposite direction, isreflected by the galvanometric mirror 1306, the fixed mirror 1305, andthe galvanometric mirror 1304. The light beam then goes through thedichroic mirror 1303, and is focused by the lens 1309 and the fixedmirror 1310 at a luminous point FEO located on the pinhole 1311 placedin an image plane P1. The light beam passes through the pinhole 1311 toobtain a beam to be detected FD which is reflected by the mirror 1312,collimated by the lens 1313, reflected by the mirror 1314, then by thesecond face of the galvanometric mirror 1304. The beam to be detected isthen reflected by the mirror 1315 and then by the second face of thegalvanometric mirror 1306. The beam to be detected FD is then focused bythe lens 1316 at a point to be detected FDO located on a point of theimage plane P2 in which is located the CCD sensor 1317 fixed to thecamera 1318. As previously, the CCD sensor can be replaced by aneyepiece allowing direct observation of the plane P2. In thisembodiment, represented in FIG. 5, the pinhole 1311 forms the firstspatial filtering system of the luminous point coming from the focusingof the light beam.

The overall operating principle is the same as in the previousdescription, namely that the galvanometric mirrors are used to scan theobject along two dimensions and the image forming on the plane P2 isrecorded by the CCD 1317.

Each point of the CCD 1317 corresponds, as in the embodiment of FIG. 4,to a unique point of the sample 1308 and, when the sample 1308 isscanned by means of the galvanometric mirrors, an image of this sampleis formed on the CCD 1317. On the other hand, in the present case, thegeometrical image in the plane P2, of a fixed point of the observedplane of the observed object, is not fixed. This has no troublesomeconsequences to the extent that a single illuminating point scans theobject.

Multipoint Laser Embodiment

This embodiment is represented in FIG. 6. It constitutes an efficientembodiment in terms of image quality and speed. Indeed:

-   -   Use of multipoint laser illumination makes it possible, in        particular on zones of small dimensions, to use an intense        illumination without saturating the sample;    -   Use of a pinhole array, which filters the beam and limits        difficulties related to possible spurious reflections.

The illuminating beam FX coming from a 100 passes through a beamattenuator 140 which may be, for example, electro-optical oracoustico-optical. It then passes through a beam expander comprising,for example, the lenses 101 and 102. The illuminating beam FX thenpasses through means for splitting said illuminating beam FX into aplurality of illuminating sub-beams. This splitting means consist of amicrolens array 103.

Solid lines have been used to represent the sub-beam coming from one ofthese microlenses and dotted lines for the sub-beam coming from anothermicrolens. Directional arrows indicate the illuminating or excitationbeam FX and the light beam retransmitted by the fluorescent object FE.

The plurality of illuminating sub-beams coming from the microlens array103 then passes through the dichroic mirror 104 and the tube lens 108and is then reflected by a mirror 109. These illuminating sub-beams arethen reflected by a rotatably mobile galvanometric mirror 110 rotatingaround an axis located in the intersection of the plane of the figureand the plane of the mirror 110, by a mirror 111, and by a rotatablymobile galvanometric mirror 112 rotating around an axis orthogonal tothe plane of the figure. They pass through the microscope objective 113and reach the observed object 114. The objective 113 is an objectiveforming an image of the observed sample at infinity. The focusing pointof each illuminating sub-beam coming from the microlens array 103 islocated in an observed plane of the observed object 114.

Preferably, the image focal plane of the objective 113 is in the objectfocal plane (in the objective-to-lens (108) direction) of the lens 108and the array of holes 160 is in the focal plane of the lens 108. Thismakes it possible to maximize the useful aperture of each sub-beam.

The illuminated object or more precisely the plurality of illuminatedpoints of the object send back a plurality of noncoherent lightsub-beams. These light sub-beams pass through the objective 113, arereflected by the galvanometric mirror 112, the mirror 111, thegalvanometric mirror 110 and the mirror 109. They pass through the tubelens 108, are reflected by the dichroic mirror 104, and then passthrough the pinhole array 130 located in the first image plane P1 andforming the first spatial filtering system. The pinhole array 130 is,for example, of the type represented schematically in FIG. 7. This array130 may be obtained by depositing a reflecting layer on a transparentglass using the “lithographic” method, the holes then beinginterruptions of the reflecting layer. In this case, a neutral filterplaced at the output of the laser can be used to attenuate laser lightreturn effects. The pinhole array can also be made up of a groundmetallic plate in which the holes are pierced by means of a laser. Thissolution overcomes laser beam return problems. The pitch (distancebetween adjacent pinhole centers) of the pinhole array 130 is the sameas the microlens array pitch 103 (distance between two adjacentmicrolens centers). The arrays 103 and 130 are positioned so that eachhole of the array 130 is the image of a point of the object on which isfocused one of the illuminating sub-beams coming from a microlens of thearray 103. This implies that the array 130 is in a focal plane of thetube lens 108, and that the focusing plane 160 of the illuminatingsub-beams coming from the microlens array 103 is also in a focal planeof the tube lens 108. The light sub-beams FE pass through the array 130to obtain sub-beams to be detected FD which are then reflected by themirror 115. The sub-beams to be detected FD are then reflectedsuccessively by the mirrors 143, 144, 145, 146, 147 constituting theassembly 142 represented by a block in FIG. 6, which are shown in FIG. 8in a view along the direction V indicated in FIG. 6. The assembly 142 isused to invert the angle of the sub-beams to be detected in relation toa plane containing the optical axis and located in the plane of FIG. 6.The direction P indicated in FIG. 8 shows the observation direction onwhich FIG. 6 is based. The sub-beams to be detected then pass throughthe lens 116 identical to the tube lens 108, and whose object focalplane is on the pinhole array 130. They are reflected by the second faceof the galvanometric mirror 110, by the mirror 117, and by the secondface of the galvanometric mirror 112. They pass through themonochromatic filter 141. They then pass through the lens 118 whichforms, in its image focal plane, the image of the array 130, and henceof the observed sample. They reach the image focal plane of the lens118, which forms the second image plane P2, in a plurality of points tobe detected. A CCD sensor 119 can be placed in this plane, whichcorresponds to the second image plane P2; however, it is also possibleto observe directly the image formed in this plane by means of aneyepiece. The focal length of the lens 116 must be equal exactly to thefocal length of the lens 108, and to allow precise adjustment, thelenses 116 and 108 may be replaced by lens doublets, the adjustment ofthe interlens distance of a doublet allowing an adjustment of the focallength of the doublet.

The inversion of the angle of the sub-beams to be detected, which isperformed by the set of mirrors 142, enables the geometrical image inthe plane P2 of a fixed geometrical point of the observed object, to bea fixed geometrical point. As an array of illuminating points is used,this condition is indispensable for the formation of a good-qualityimage.

FIG. 9 shows the principle of laser beam focusing by the microlens array103. The value of the angle α indicated in this figure is preferably

$\alpha = {{ouv}\;{\frac{F_{113}}{F_{108}}.}}$The width of the Airy disk produced in the plane 160 is then

$L_{160} = {1,22\;\frac{\lambda_{las}}{ouv}\frac{F_{108}}{F_{113}}}$where F₁₀₈ is the focal length of the lens 108, F₁₁₃ is the focal lengthof the objective 113, ouv is the numerical aperture of the objective113, λ_(las) is the wavelength of the laser. The distance D between twoadjacent microlenses is preferably at least 10 times the width of eachhole. The microlenses of the array 103 are spherical microlenses limitedby squares and adjacent to each other. The width of each microlens (sideof square limiting it or distance between centers of two adjacentmicrolenses) is equal to the distance D between two adjacent pinholes ofthe array 130, and the diameter L₁₃₀ of a pinhole is equal, for example,to the width of the Airy disk produced in the plane 160, or to half thiswidth. The focal length F₁₀₃ of each microlens of the microlens array103 and its width D are moreover related by the relationship

${\frac{D}{2F_{103}} = {\alpha = {{ouv}\;\frac{F_{113}}{F_{108}}}}},$so that

$F_{103} = {\frac{D}{2}\frac{F_{108}}{F_{113}}{\frac{1}{ouv}.}}$For example we may have:

-   F₁₁₃=2 mm-   ouv=1.25-   F₁₀₈=200 mm-   D=2 mm-   λ_(las)=488 nm (argon laser)-   F₁₀₃=80 mm-   L₁₃₀=23 μm

For maximum resolution, with pinholes having a diameter sufficientlysmaller than the Airy disk, and to enable optimum subsequentdeconvolution of the confocal image, the sampling pitch P₁₁₉ on the CCDsensor (distance between centers of two adjacent pixels) must agreewith:

$P_{119} = {\frac{1}{4{ouv}}\frac{F_{118}}{F_{113}}\frac{\lambda_{las}\lambda_{fluo}}{\lambda_{las} + \lambda_{fluo}}}$${{so}\mspace{14mu}{that}\mspace{14mu} F_{118}} = {4{ouv}\; P_{119}F_{113}\frac{\lambda_{las} + \lambda_{fluo}}{\lambda_{las}\lambda_{fluo}}}$For example, with P₁₁₉=12 μm and still in the same dimensioning example,we obtain:F₁₁₈=477 mmFor maximum resolution but without subsequent deconvolution of theconfocal image, it is possible to accept:

$F_{118} = {{\frac{4}{2\sqrt{2}}{ouv}\; P_{119}F_{113}\frac{\lambda_{las} + \lambda_{fluo}}{\lambda_{las}\lambda_{fluo}}\mspace{14mu}{so}\mspace{14mu}{that}\mspace{14mu} F_{118}} = {168\mspace{14mu}{mm}}}$With pinholes whose diameter is an Airy disk, the following may beacceptable:

$F_{118} = {\frac{4}{2\sqrt{2}}{ouv}\; P_{119}F_{113}\frac{\lambda_{las} + \lambda_{fluo}}{\lambda_{las}\lambda_{fluo}}}$

It is thus useful to be able to modify the focal length of the lens 118,or replace it with a zoom-type system or with a variable magnificationsystem using interchangeable optical elements.

The galvanometric mirrors 110 and 112 are controlled so as to move theilluminated points in the observed object 114, as shown in FIG. 10. FIG.10 shows, shaded, all the points illuminated by the excitation sub-beamsFX in the observed plane of the object, for a reference position of thegalvanometric mirrors. The diameter of the points represents theapproximate diameter of the corresponding diffraction disks. The line301 superposed on the drawing shows the path followed by an illuminatedpoint 300 when the galvanometric mirrors are operated. This path is runalternately in both directions. When this path is followed, eachilluminated point scans a small part of the image plane, and all theilluminated points scan the entire image plane. A confocal image of theentire observed plane of the object 114 is thus generated on the sensor119. The contour 302 shows the limit of the useful zone, from which agood-quality confocal image is generated on the sensor. Many variants inthe path covered may be used, the essential requirement being that theentire useful zone should be scanned, two distinct parts of the usefulzone generally being scanned by distinct illuminated points.

The beam attenuator 140 must be controlled according to the position ofthe galvanometric mirrors and their speed. These parameters may beobtained in a known manner by feedback from galvanometers, or may beobtained without feedback through a galvanometer control system with alower accuracy. Ilas is used to denote the intensity of the laser beamafter passing through the attenuator 140, and Vscan the scanning speed,i.e. the movement speed in the observed plane and along the x axis of anilluminated point in the observed object. This scanning speed is dueonly to the fastest galvanometric mirror. The position of thegalvanometric mirrors may be characterized by the position of theilluminated point in the object 114. FIG. 12 illustrate the value ofratio Ilas/Vscan as a function of the position of the illuminated pointin the sample 114. FIG. 11 represents a set of illuminated points,including the point 300 also shown in FIG. 10, for the referenceposition of the galvanometric mirrors. In this figure is represented inunbroken lines the edges of the surface representing the functionIlas/Vscan as a function of the coordinates x, y of the point 300 in theobserved plane. In FIG. 12 is represented the shape of this functionalong the line 310 of FIG. 11. A constant value of Ilas/Vscan over theentire trajectory, i.e. a control of the attenuator as a function ofonly the scanning speed, would make it possible in principle toeliminate illumination variations due to variations in the scanningspeed. However, the curve of FIG. 12 also allows the attenuation of theeffects of an uncontrolled variation in the oscillation amplitude of themirrors, however to the detriment of luminous intensity. It is alsopossible to have the beam attenuator operate in binary mode. Intensitycontrol then takes place for example in accordance with FIG. 13. Onlythe central part of the trajectory on which the speed is roughlyconstant is used. Finally, it is also possible not to use any beamattenuator. The image remains of acceptable quality but may be affectedby local intensity variations, which may be compensated by subsequentdigital processing. In general, the higher the number of pinholes thegreater the surface area of an illuminated point, and the smaller theillumination intensity variations. A sufficiently dense pinhole arraycan thus partially replace a beam attenuator.

To each utilizable objective 113, characterized by its aperture, itsmagnification and the position of its image focal plane, therecorresponds an optimum dimensioning of the pinholes used, of themicrolenses and of the lens 108. These elements can be designed so thatthey suit all the objectives, but the properties of the system will besub-optimal with certain objectives. It is also possible to consider aseries of objectives designed to give the best results with a givendimensioning of the rest of the system. However, it is preferable to beable to change the pinholes used and the microlenses so as to be able tooptimize them according to the objectives used, the excitation andfluorescence wavelengths, the fluorescence level, the desiredacquisition speed, and the desired resolution. Accordingly, themicrolens array 103, the pinhole array 130 and the dichroic mirror 104can be made integral with each other and form an exchangeable unit in asingle piece. This avoids the user having to adjust the alignment of thedifferent elements: alignment problems are settled when the unit ismanufactured. The positioning of the unit in the rest of the systemrequires only a sufficient angular accuracy (of the order of 1milliradian).

The present embodiment may be combined with all known imaging modes inconfocal microscopy. In particular, the microscope stage may be equippedwith a piezoelectric or step-motor type vertical movement system so asto be able to generate three-dimensional images by modifying thefocusing plane. Systems based on acoustico-optical attenuators may beused to switch several lasers and excite different fluorophores, so asto generate, by superposition, images with a richer information content.The system is also compatible with the use of the two-photon method, thenumber of pinholes then needing to be adjusted so that a sufficientintensity remains available on each focusing point of the beam. Filterwheels (filter 141) can be used to modify detected wavelengths. Thedichroic mirror 104 can be replaced by a semi-transparent mirror toenable the changing of excitation wavelengths and fluorescence byswitching a laser and changing the filter 141, without having toexchange the dichroic mirror 104. Replacement of the dichroic mirror 104by a semi-transparent mirror also allows the device to be used toobserve nonfluorescent diffractive samples in reflection.

It is possible, by means of relay lenses, to modify the device so thatthe entire scanning system is placed at the back of an intermediateimage plane. Such a solution can be useful in designing a scanningsystem adaptable to any type of microscope.

It is of course possible to use only one pinhole, in which case thespeed characteristics are those of a confocal scanning microscope ofcurrent design with galvanometric mirrors. However, the advantage of thesystem is the possibility of displaying the image directly and recordingon a camera.

It may also be useful to equip the system with a device for positioningthe sample 114 in the direction of the optical axis. This in fact makesit possible to obtain three-dimensional images made up of series oftwo-dimensional images each obtained at a different depth. Thethree-dimensional image obtained can then undergo three-dimensionaldeconvolution which improves its resolution. Prior to deconvolution, thepoint spread function (PSF), or three-dimensional impulse response, mustbe measured, for example on a fluorescent microbead.

Laser Multipoint Embodiment with Compensation of Certain Defects.

FIG. 14 illustrates an embodiment derived from the preceding one butimproved by several additional devices.

The plurality of illuminating sub-beams coming from the microlens array103 passes through a rotatably mobile glass 106 rotating around an axis107, then an optional lens 135, then a second spatial filtering systemmade up of a pinhole array 105, and only then reaches the dichroicmirror 104.

The surface of the glass 106 is divided into a set of sub-surfaces, forexample the sub-surfaces 120 and 121. Half of these sub-surfaces have anextra thickness generating a phase shift of 180 degrees in theilluminating sub-beams passing through them. The sub-surfaces havingextra thicknesses are distributed in a pseudo-random manner in all thesub-surfaces. Each sub-surface is approximately square. The width of theside of the square is equal to the distance between two adjacentpinholes in the array 105. The glass 106 is positioned so that eachpinhole of the array 105 is placed under a difference sub-surface. Thefast rotation of the glass 106 allows the generation of pseudo-randomphase shifts in all the illuminating sub-beams, so that the spatialcoherence of the sub-beams is broken after passing through the glass106. In fact, the coherence between beams is capable of disturbing theimage slightly.

A practical difficulty has to do with the fact that objectives withdifferent magnifications have distinct image focal planes. Consequently,the object focal plane of the lens 108 (in the direction from theobjective to the lens 108) can be the same as the image focal plane ofthe objective only for one of the objectives used. This difficulty maybe thereby decreasing the available luminous intensity. The lens 135enables this difficulty to be overcome without any loss in luminousintensity. It must be dimensioned so that a plane wave in the object hasas its image a plane wave between the lens 135 and the microlens array103. Changing an objective then requires the changing of the lens 135.

The pinhole array 105 forming the second spatial filtering system isdesigned to filter the laser sub-beams coming from the microlenses ofthe array 103. This makes it possible to overcome any imperfections inthese microlenses. Each pinhole of the array 105 is placed at the pointof focus of a corresponding microlens and its diameter may, for example,be an Airy disk or an Airy half-disk, depending on whether one wishes tooptimize resolution or brightness.

The microlens array 131 placed before the first spatial filtering systemmade up of the pinhole array 130 is especially useful when theexcitation wavelength is much smaller than the fluorescent emissionwavelength and when the diameter of the pinholes is of the order of anAiry disk. It then allows a slight improvement in resolution withoutsacrificing brightness.

FIG. 15 shows the operating principle of the microlenses of the array131 and the pinholes of the array 130. As indicated in this figure, andin FIG. 9, the angle

$\alpha = {{ouv}\;{\frac{F_{113}}{F_{108}}.}}$The focal plane 132 of the lens 108 has been represented. In the absenceof the microlens array 131, the pinhole array 130 must be placed in thisplane, and the divergence angle of the beam after passing through thepinholes, to the extent that these holes are sufficiently large, wouldbe α. What makes the lens array 131 useful is that it increases thisdivergence angle, which is equivalent to reducing the diameter of theAiry disk formed on each hole of the pinhole array 130. This makes itpossible to reduce the diameter of the pinholes of the array 130 withoutany loss in luminous intensity, thus subsequently allowing reception ona CCD sensor without the resolution being decreased by the superpositionof the Airy disks obtained for close points. The divergence angle of thebeam after passing through the pinholes becomes, because of themicrolenses of the array 131, β=mα with, typically, m=1.5 to m=4. Thedistances d₂ between the microlens array 131 and the pinhole array 130,d₁ between the microlens array 131 and the focal plane 132 of the lens108, and the focal length F₁₃₁ of the microlenses of the array 131, arelinked by the thin lens equation (which may be modified if necessary totake into account the thickness of the lenses):

${\frac{1}{d_{2}} - \frac{1}{d_{1}}} = \frac{1}{F_{131}}$and we moreover have, given the desired coefficient m:

$\frac{d_{1}}{d_{2}} = m$Furthermore, for reasons of overall dimensions, we have:D=2d₁caαwhere c≧1. Typically it is possible to take c=2.Given also the expression of the angle

${\alpha = {{ouv}\;\frac{F_{113}}{F_{108}}}},$we finally obtain:

$d_{1} = {\frac{D}{2c}\frac{F_{108}}{F_{113}}\frac{1}{ouv}}$$F_{131} = {\frac{1}{m - 1}d_{1}}$ $d_{2} = {\frac{1}{m}d_{1}}$

The width of the pinholes on the array 130 can be equal to the diameterof the Airy disk on these holes, that is:

${L_{130} = 1},{22\;\frac{1}{m}\frac{\lambda_{fluo}}{ouv}\frac{F_{108}}{F_{113}}}$where λ_(fluo) is the wavelength corresponding to maximum fluorescentemission.Using the dimensioning example used in the previous embodiment for thearrays 103 and 105 we obtain, for c=2 and m=4, and for λ_(fluo)=518 nm(fluoresceine):

-   F₁₁₃=2 mm-   ouv=1.25-   F₁₀₈=200 mm-   D=2 mm-   F₁₀₃=80 mm-   d₁=40 mm-   F₁₃₁=13.3 mm-   d₂=10 mm-   L₁₃₀=12 μm    Multipoint Embodiment with Incoherent Lighting

This embodiment represented in FIG. 16 differs from the one representedin FIG. 6 by the fact that noncoherent illumination is used. This limitsthe imaging speed but allows the use of less costly illumination inwhich the wavelength can easily be adjusted, or the use of white lightillumination for observation under reflected light.

The illuminating beam FX is supplied, for example, by the incandescentarc 150 of a mercury vapor or Xenon lamp. This illuminating beam passesthrough a collector 151, a field diaphragm 152, and a monochromaticfilter 153 selecting the fluorescence excitation wavelength of theobject to be observed. Then this illuminating beam FX passes through thelens 154, the aperture diaphragm 155, the lens 156, and reaches directlythe pinhole array 105 forming the second spatial filtering system. Thesystem made up of elements 150 to 156 constitutes Köhler illuminationand can be replaced by any other Köhler illumination system. The rest ofthe system is the same as in the embodiment of FIG. 6.

A great part of the available luminous intensity is reflected by thepinhole array 105, thus limiting the brightness of the image formed inthe plane 119 corresponding to the second image plane P2. In order forthe acquisition time and the brightness of the image to remain withinreasonable limits, it is preferable to use an array 105 consisting ofpinholes very close to each other. While in the preceding embodiment,the distance between two pinholes can typically be about 20 times thewidth of each hole, in the present embodiment it is preferable to limitthis distance, which may be for example from 2 to 4 times the width ofthe pinhole. This generates image disturbances, but the confocal effectis maintained and the disturbances may be eliminated for example bythree-dimensional deconvolution if a three-dimensional image isacquired, composed of a series of two-dimensional images acquired atdifferent depths in the object.

To allow the exchange of two pinhole arrays without this exchangerequiring complex adjustment, and to reduce manufacturing cost, thepinhole arrays 105, 130 and the dichroic mirror 104 can be incorporatedin a single easily-made element, represented in FIG. 17. The dichroicmirror 400 is integral with a transparent cube 401. Glasses 402, 403 aremounted on the sides of the cube, to which they are secured by suitableparts, for example 404. Empty spaces 405, 406 are left between theglasses and the cube 401. The side of the glasses 402, 404 which facesthe cube is covered with a thin metallic layer and a thin photosensitiveresin layer. The pinholes are obtained by insolating the photosensitiveresin by means of a white light projector. The shape of the beam comingfrom this projector has been represented symbolically by a dotted line.The projector is focused for example on the plane made up of the face ofthe glass 402 which carries the thin layers. Each illuminated point ofthe glass 402 will become a pinhole. Because of the configuration of thesystem, the points of the glass 403 which are illuminated are correctlyplaced without any additional adjustment being necessary, and willbecome the pinholes corresponding to the holes in the glass 402. Afterinsolation, an appropriate liquid is introduced into the empty spaces405, 406 so as to remove the resin at the insolated locations, then anacid is used to remove the metal at these same points. The pinholes arethen obtained. A solvent can be used to remove the residual resin layer,and then the whole thing can be cleaned. Finally, it may be useful tointroduce an optical liquid or a transparent plastic in the empty space,preferably with the same index as the plates 402, 403. This preventsunnecessary reflections on the contact surfaces. If a material with thesame index as the plates 402, 403 is introduced into the empty spaces,it is then possible to use in the overall design glasses 402, 403 groundon the side facing the cube. This allows better dispersion of incidentlight by the reflecting parts. The monochromatic filters 153 and 141 canalso be incorporated in this cube, thus making it possible to modify theimaging mode by exchanging only one component. If observation underreflected light is desired, the dichroic mirror can be replaced by aneutral separator cube.

Another solution simplifying the exchange of pinhole arrays consists inusing only one hole array which is traversed in one direction by theilluminating beam FX and in the other direction by the light beam FEemitted by fluorescence from the observed sample. In this case, thesystem is modified as indicated in FIG. 18: a single pinhole array 105used, but the dichroic mirror 104 is placed between the lens 156 and thepinhole array 105. The microlens array 131 is also eliminated and themechanical dimensioning is modified to take into account the replacementof the array 130 by the array 105. This configuration is particularlysimplified, but one consequence of this simplification is the presenceof a high light intensity resulting from partial reflection of theilluminating beam FX by the hole array 105 (stray light), which must beeliminated by the dichroic mirror 104 and the filter 141. The advantageof this configuration is that the exchange of the array 105, whichconstitutes both the means for splitting the illuminating beam intosub-beams, and the first spatial filtering device, can take placewithout alignment problems.

It is also possible to eliminate one of the galvanometric mirrors 110and 112 and replace it by a fixed mirror, provided a slight modificationis made in the axis of rotation of the remaining mirror and its controlso as to have, for each illuminated point, a sufficiently longtrajectory. FIG. 19, prepared using the same conventions as FIG. 10,shows the trajectory 500 of an illuminated point 503 in the observedplane of the object 114, as well as the zone 502 in which a good-qualityimage is obtained, the position of the illuminated points for thereference position of the remaining galvanometric mirror being asindicated in the figure, limited by the contour 501. A rectilineartrajectory 500 slightly oblique in relation to the director vectors x, yof the two-dimensional periodic array of pinholes makes it possible toscan the entire plane without requiring a second galvanometric mirror.It is all the more easy to obtain an extended zone 502 as theilluminated points are close to each other. As it is preferable in thepresent embodiment, in order to maximize the illuminating intensity, tohave many illuminated points, the present embodiment is particularlywell suited to the use of this technique. However, it is also possibleto use a rectilinear trajectory in an embodiment using a microlensarray, provided the illuminated points are sufficiently numerous.

Similarly, it is possible to use the two galvanometric mirrorscontrolled in quadrature so that the trajectory of the image of a point504 moves on a circle 505, and to modify progressively the diameter ofthis circle so that the trajectory covered during an image acquisitionis a spiral. If the pinholes are sufficiently numerous and close to eachother the entire plane is scanned. The advantage of this solution isthat the movement of the illuminated point is at roughly constant speed;however, this type of scanning tends to generate over-illumination onlines parallel to the director vectors of the pinhole array.

This embodiment has the advantage of not requiring a laser beam. It alsohas the advantage of permitting easy modification of excitation andfluorescence emission wavelengths in the entire visible and UV domain.On the other hand, the low illumination limits the imaging speed.

This second embodiment may be combined with the first in the samesystem, shutters being utilizable for switching from one illuminatingmode to another.

Embodiment with an Additional Reflection of the Illuminating Beam on theMobile Mirror

FIG. 20 illustrates another embodiment, which offers the advantage oflow cost. It uses noncoherent illumination and requires feweradjustments or specific elements than the preceding embodiment. It usesonly one galvanometric mirror and a single face of this mirror, and usesan array of microscopic mirrors.

The excitation beam FX is a noncoherent beam produced for example by aXenon arc lamp equipped with a collector and an optical system allowingthe generation of Kohler illumination. It is filtered by a narrowbandpass filter which selects the excitation wavelength of thefluorescence. It reaches the scanning device at 600. The illuminating orexcitation beam FX is reflected by the dichroic mirror 601 then by thepolarizing beam splitter 602. The illuminating beam FX is then reflectedby the galvanometric mirror 603, passes through the quarter-wave plate604 whose neutral axis is 45 degrees from the plane of the figure,passes through the lens 605 and reaches the microscopic mirror array 606placed in the image focal plane of the lens 605. The microscopic mirrorarray 606 is of the type represented in FIG. 21 and consists, forexample, of a set of microscopic mirrors (for example 650) forming asquare matrix on a transparent glass with antireflection treatment. Themicroscopic mirror array can also have a hexagonal grid, thus improvingthe efficiency of the system. Microscopic mirrors can be obtained forexample by lithography, each mirror being composed of a reflecting layerdeposited on the glass 606, the glass being preferably antireflectiontreated. The reflecting layer can for example be an aluminum layer. Itis also possible to use mirrors composed of alternating layers ofdifferent indices, and which can thus be selective in terms ofwavelength. The part of the illuminating beam FX which is reflected bythe microscopic mirrors is in the form of a plurality of illuminatingsub-beams FX2. These illuminating sub-beams FX2 pass again through thelens 605, the quarter-wave plate 604, and are reflected by thegalvanometric mirror 603. The illuminating sub-beams FX2 pass throughthe polarizing beam splitter 602 then the lens 608 and the tube lens609. They pass through the objective 610 and are focused in a pluralityof illuminating points FXO on the observed fluorescent object 611. Thelight sub-beams FE retransmitted by fluorescence of the illuminatedpoints of the fluorescent object pass through the objective 610, thetube lens 609, the lens 608 and the polarizing beam splitter 602. Thelight sub-beams FE are reflected by the galvanometric mirror 603, passthrough the quarter-wave plate 604 and the lens 605, and reach themicroscopic mirror array 606 which then constitutes the first spatialfiltering system. The part of the light sub-beams FE which are reflectedby this microscopic mirror array constitutes the plurality of sub-beamsto be detected FD. These sub-beams to be detected FD pass through thelens 605, the quarter

wave plate 604, are reflected by the galvanometric mirror 603, also arereflected by the polarizing beam splitter 602, pass through the dichroicmirror 601 and the lens 612, and then reach the CCD sensor 613 locatedin the image focal plane of the lens 612 which coincides with the secondimage plane P2. The image formed in the plane in which the sensor 613 islocated can also be observed by means of an eyepiece.

The focal plane 607 of the tube lens 609 is also a focal plane of thelens 608. The other focal plane of the tube lens 609 is preferably thesame as the image focal plane of the objective 610. Preferably, thegalvanometric mirror 603 is in a common focal plane of the lenses 605,608 and 612. The microscopic mirror array 606 is in a focal plane of thelens 605 coinciding with the first image plane P1. The CCD sensor 613 isin a focal plane of the lens 612. For example, the lenses 609, 608, 605and 612 can have a focal length of 200 mm, the objective 610 can be aimmersion-type Nikon objective x100 with an aperture of 1.4. In thiscase, the array 606 can for example be made up of microscopic mirrorswhose diameter is 30 microns, the distance between two adjacent pointsbeing for example 150 microns. The dichroic mirror 601 reflects thewavelength of the excitation beam and allows the passage of thefluorescent emission wavelength. The retardation plate 604 is used tomodify the polarization of the beams so that, for example the pluralityof sub-beams to be detected FD are reflected by the polarizing splitter602 whereas the plurality of light sub-beams FE pass through it.

The galvanometric mirror 603 can for example be rotatably mobile aroundan axis located in the plane of the figure. In this case, themicroscopic mirror array 606 must be oriented so that the trajectory ofan illuminated point in the object is slightly oblique in relation to adirector vector of the periodic array of illuminated points, asdescribed by the trajectory 500 in FIG. 19, thus allowing the scanningof the entire object by means of a single galvanometric mirror.

The microscopic mirror array 606 is placed in a plane P1 whichconstitutes the first image plane, in which is placed the first spatialfiltering system. The mirror array 606 forms both the first spatialfiltering system, which filters the light beam FX coming from the sampleto give the beam to be detected FD, and the second spatial filteringsystem, which splits the illuminating beam FX into a plurality ofsub-beams FX2.

As in the first embodiment, it would have been possible to place thegalvanometric mirror 603 in the image focal plane of the objective ornear this place. The relay lenses 609 and 610 have been introduced toillustrate the possibility of such a design, which lends itselfparticularly to the setup of a scanning device that can be used with anytype of microscope.

The position of the array 606 must be adjusted in rotation around theoptical axis so that a director vector of the illuminated point array inthe observed object is slightly oblique in relation to the trajectory500 of an illuminated point when the mirror 603 rotates, as indicated inFIG. 19. The position of the array 606 must be adjusted in translationin the direction of the optical axis so that the array 606 is conjugateto the CCD 613. These adjustments are easily carried out.

The present embodiment offers the advantage of being low in cost andrequiring few adjustments. It is well suited to a low-cost version ofthe invention, intended for fluorescence observation. The device an beequipped with a vertical sample movement system, in which case theobtained image quality can be improved by three-dimensionaldeconvolution. Such deconvolution makes it possible to improveresolution and to eliminate most defects due to stray reflections in thesystem, and possibly to also compensate for defects related to limiteddistance between the points of the array 606. It requires priormeasurement of the point spread function (PSF) which may be carried outby imaging a fluorescent microbead using a known technique.

The confocal scanning device can comprise all the elements contained inthe zone limited by the dotted line 614. In this case the user mustsimply connect to the system a microscope and an eyepiece or a camera.However, the confocal scanning device can also include only the elementsincluded in the zone limited by the dotted line 615. In this case, theuser is free to choose the lenses 608 and 612, thus enabling him/her forexample to modify the magnification of the system by an appropriatechoice of the lens 612. It is also possible to add a lens in the plane607 in order to bring to infinity the image focal plane of themicroscope in the case in which it is not exactly placed in a focalplane of the tube lens 609.

To obviate the use of a polarizing beam splitter and prevent the loss ofluminous intensity resulting therefrom, the device may be modified asindicated in FIG. 22. In FIG. 22 the various elements of the device arethe same as in FIG. 20, the elements 602, 603 and 605 however beingreplaced respectively by the elements 602 b, 603 b and 605 b. Thepolarizing mirror 602 is replaced by the simple mirror 602 b on whichare reflected the illuminating beam FX and the plurality of sub-beams tobe detected FD. The galvanometric mirror 603 b is roughly twice as longas the mirror 603, and the lens 605 b is also larger than the lens 605.The optical axis of the lens 605 b is offset in relation to the opticalaxis of the illuminating beam FX coming from the galvanometric mirror,so that the illuminating beam FX comes in on a slant on to themicroscopic mirror array 606. The illuminating sub-beams FX2 reflectedby the array 606 then reach a zone of the mirror 603 b distinct fromthat which was illuminated by the illuminating beam FX. After reflectionon 603 b, the illuminating sub-beams FX2 pass next to the mirror 602 b.The plurality of light sub-beams FE coming from the sample follow thereverse path so that, after reflection on the pinhole array, thesub-beams to be detected FD reach the sensor. The device in FIG. 22, forits design simplicity and its effectiveness, constitutes the preferredembodiment for fluorescence observation with incoherent illumination.

An advantage of the present embodiment is that the illuminating beam FXis reflected a first time on a galvanometric mirror before reaching themicroscopic mirror array. It is then reflected a second time (beams FX2)before reaching the observed object. Consequently, the zone illuminatedby the illuminating beam on the microscopic mirror array moves, whereasthe illuminated zone in the observed object remains stationary. Thisdiffers from the preceding multipoint embodiments in which theilluminated zone in the image plane moves, and hence the illuminatedzone in the observed object cannot be made stationary unless it islimited by a diaphragm located in the image plane, causing an energyloss, or by moving the illuminating points over very short trajectories,which resolves the problem only partially and poses synchronizationproblems.

The synchronization of the system can be simplified by using themovement of the illuminated zone over the microscopic mirror array. Whenthe illuminating beam, moving over the microscopic mirror array, leavesthe covered zone of microscopic mirrors, the observed object is nolonger illuminated. One need only have extinction coincide with the endpositions of the galvanometric mirror in order to eliminate illuminationnonhomogeneity caused by the slowing of mirror movement around the end(stationary) points of their trajectory.

FIG. 23 illustrates this operating principle. FIG. 23( a) shows theplate 606 on which is located a microscopic mirror array 1601. Alsorepresented is the zone 1602 illuminated by the illuminating beam whenthe galvanometric mirror 603 reaches an end position of its oscillatorymovement. In the position indicated in FIG. 23( a) the illuminated zoneis outside the zone covered with microscopic mirrors, the illuminatingbeam thus passes through the plate without being reflected, and theobserved object is not illuminated. In this position, the plate 606consequently makes it possible to eliminate the illuminating beam beforeit reaches the observed object. The zone 1602 moves over the plate 1600but, in the end position represented in FIG. 23( a), its movement speedis cancelled before the movement of the illuminated zone resumes in thedirection indicated by the arrow 1603. The illuminated zone 1602 thenpasses through the zone 1601 covered with microscopic mirrors, at aroughly constant speed, as indicated in FIGS. 23( b) to 23(f). Duringthis phase, the observed object is illuminated. When the position of themobile mirror reaches the extreme reverse of its oscillatory movement,the illuminated zone leaves the zone covered with microscopic mirrorsand its speed is cancelled, as indicated in FIG. 23( g). The observedobject again ceases to be illuminated. The illuminated zone 1602 thenmoves again in the opposite direction for another crossing of the zone1601 covered with microscopic mirrors. If a camera is used for signaldetection, the movement of the galvanometric mirror can be synchronizedwith acquisition by the camera. During the camera exposure time, theilluminated zone 1602 can, for example, carry out one or severalcomplete crossings of the zone 1601.

In FIG. 23 has been indicated a particular microscopic mirror 1604. FIG.24 illustrates the movement in the object of the geometrical image 1702of the mirror 1604, and hence of the corresponding illuminated point inthe object. The mirror 1604 may be assimilated with a point of the firstimage plane P1, in which is located the glass 606. FIG. 24( b)represents the position of the geometrical image 1702 of the microscopicmirror 1604, as well as the observed zone 1701 which is the geometricalimage of the illuminated zone 1602 on the plate 606, and this for aposition of the illuminated zone 1602 on the plate 606 which is the onerepresented in FIG. 23( b). The point 1702 moves along the directionindicated by the arrow but has not yet reached the zone 1701, and ishence not yet illuminated. This reflects the fact that in FIG. 23( b)the illuminated zone 1602 has not yet reached the point 1604. The point1702 then reaches the observed zone 1701 as indicated in FIG. 24( c) andhence begins to be illuminated, this reflecting the fact that theilluminated zone 1602 reaches the point 1604 as indicated in FIG. 23(c). The point 1702 is then illuminated and passes through the zone 1701at constant speed, as indicated in FIG. 24( d), which reflects the factthat the point 1604 is in the illuminated zone as indicated in FIG. 23(d). The point 1702 reaches the limit of the observed zone as indicatedin FIG. 24( e), which reflects the fact that the point 1604 is at thelimit of the illuminated zone 1602, as indicated in FIG. 23( e). Thepoint 1702 then comes out of the observed zone and is then no longerilluminated, as indicated in FIG. 24( f), which reflects the fact thatthe illuminated zone 1602 has gone beyond the point 1604, as indicatedin FIG. 23( f). The point 1702 then continues to move at constant speedand then its speed is cancelled and it changes direction to come backtowards the observed zone. When the movement speed of the point 1702 iscancelled, the movement speed of the illuminated zone 1602 on the plate606 is also cancelled, and this zone is hence in one of the positionsillustrated in FIGS. 23( a) and 23(g). When the movement speed of thepoint 1702 is cancelled, the entire illuminating beam is thuseliminated. The plate 606 acts as a limitation device eliminating thepart of the illuminating beam which passes outside of a selectionsurface coinciding with the zone 1601 covered by the microscopic mirrorarray. When the movement speed of the point 1702 (or of any otherequivalent point) is cancelled, the entire zone 1602 illuminated by theilluminating beam is outside the selection area 1601, and theilluminating beam FE is entirely eliminated. The observed zone is thustraversed successively by a set of illuminated points each passingthrough it at constant speed. Speed variations of the illuminated pointsare effectively eliminated by this method.

FIG. 25 represents the shape of part of the array of reflectingmicroscopic mirrors 1601, in the case of a hexagonal grid array. Thedirector vectors of the array are u and v, as represented in the figure.This array includes a set of reflecting microscopic mirrors, shaded inthe figure. The line 1623 passes through the center of the microscopicmirror 1625 and is oriented in the direction of movement of theilluminated zone 1602 over the array, which is also the direction ofmovement over the array of the geometrical image of a fixed point of theobserved object. The line 1624 is directed along a director vector ofthe array and passes through the center of the microscopic mirror 1625.The microscopic mirror 1627 is the first mirror located on a line 1631orthogonal to the line 1624 and passing through the center of themicroscopic mirror 1626. The point 1630 is the intersection of the lines1624 and 1623. The following notations are used:

-   D1 the distance between the center of the mirror 1627 and the point    1630.-   D2 the distance between the center of the mirror 1626 and the center    of the mirror 1627.-   N the number of microscopic mirrors located on the line 1624 between    the points 1625 and 1626, including these points.

For the scanning conditions to be optimal it is necessary for N to besufficiently large and agree moreover with the inequality D1=D2/N. Also,as the array is extended beyond the points indicated in the figure, thetotal number of points on the line 1624 must be a multiple of N, and thelimits of the array must be orthogonal to the line 1624.

The more extensive the zone 1601 in the direction of movement of theilluminated zone 1602, the greater will be the possibility of minimizingluminous energy losses related to the nonreflection of the beam on theplate 606 at the end points of movement of the illuminated zone on theplate 606. However, this technique always causes a certain loss ofenergy. This loss may be avoided, for example, by modifying the movementof the galvanometric mirror and hence the trajectory of a geometricalpoint 1702, image of the point 1604, so that the illuminated zone 1602remains in the zone 1601 covered by the microscopic mirror array.However, if the change of direction of the point 1702 at the end pointsof its trajectory is not sufficiently rapid, this technique createsillumination nonhomogeneities in the observed object and hence avariation of the three-dimensional point spread function (PSF) accordingto the considered point. Because the microscopic mirror array isperiodic, the dependence of this PSF according to the considered pointis itself periodic. Illumination nonhomogeneities can be corrected bymeans of appropriate image processing algorithms.

Usual deconvolution algorithms consist in calculating at each point ofthe object a value:V(r ₀)=∫∫∫D(r−r ₀)I(r)d ³ rwhere r₀ is the position vector of the considered point, r is a variableposition vector, where the deconvolution function D(r−r₀) allows theinversion of the PSF, and where I(r) is the non-deconvoluted imageobtained from a series of two-dimensional confocal images each obtainedfor a different observed plane, a piezoelectric positioner along the zaxis (vertical) being used for the acquisition of such a series ofimages. This calculation can be carried out in the frequency domain bymeans of known three-dimensional Fourier transform techniques, or in thespatial domain by means of filtering techniques.

When the illuminating nonhomogeneities are not too strong, or when thediameter of the microscopic mirrors is small (less than a Airyhalf-disk), illumination nonhomogeneities can be compensated by amultiplication of the three-dimensional image I(r) by an appropriatefunction C(r), which is periodic along the same director vectors as thearray of illuminating points in the observed object, and compensatedirectly for illumination variations. This function C(r) can be obtainedfor example by observing a uniformly fluorescent flat object, anon-deconvoluted image I₀(r) of which is then obtained. We then have

${C(r)} = {\frac{1}{I_{0}(r)}.}$The multiplication of I(r) by C(r) can be followed or not by adeconvolution of the usual type.

In the more general case, the spatial dependency of the deconvolutionfunction transforms the equation describing the deconvolution into:V(r ₀)=∫∫∫D(r ₀ ^(h) ; r−r ₀)I(r)d ³ rwhere D(r₀ ^(h); r−r₀) is dependent on the horizontal component r₀ ^(h)of the vector r₀ but is periodic on this variable, so that:D(r ₀ ^(h) ; r−r ₀)=D(r ₀ ^(h) +ku+lv; r−r ₀)where u and v are the director vectors of the array of illuminatingpoints in the observed object and where k and l are arbitrary wholenumbers. The function D(r₀ ^(h); r−r₀) can be obtained on a grid of theperiodic array of illuminating points, then making it possible to obtainit over the entire object. At each point of the grid of the array, itcan be obtained by inverting, in the frequency domain (i.e. in Fourierrepresentation), a PSF measured at this point by moving a fluorescentmicrobead around the point, the value P(r) of the PSF being theintensity value measured at the measurement point when the position ofthe microbead in relation to the measurement point is defined by thevector r.

The device in FIG. 22 can be combined with a second galvanometric mirrorto be able to perform two-dimensional scanning as in the secondembodiment. The resulting layout is shown FIG. 26 in which agalvanometric mirror 615 has been added and the other elements have beenmoved accordingly. The axis of rotation of the mirror 615 is orthogonalto that of the mirror 603 b. By controlling the two mirrors inquadrature, it becomes possible to impart a circular or two-dimensionaltrajectory to each illuminated point. This trajectory is for examplesimilar to the trajectory 505 of FIG. 19. The advantage of such atrajectory is that it does not result in any energy losses. However,this trajectory generates periodic illumination nonhomogeneities, whichcan be compensated as previously by using a deconvolution functiondepending on the considered point or by multiplying the non-deconvolutedimage by an illumination nonhomogeneity compensation function.

A similar technique can be used with laser illumination, theilluminating beam then having to be reflected a first time on thegalvanometric mirrors before reaching the microlens array. Such adevice, derived from the one in FIG. 6, is represented in FIG. 27.Mirrors 162 and 161 have been added to modify the trajectory of theilluminating beam, and the laser 100 and the lens 101 have been placedupstream of the galvanometric mirrors. The mirror 115 has been replacedby a dichroic beam splitter 164. Preferably, the beam laser coming from100, represented in a thick dotted line, is focused by the lens 101 onthe galvanometric mirror 112, which is the only mobile and which is alsoin a focal plane of the lens 116. The angular movement of thegalvanometric mirror then results in the movement of the illuminatingbeam over the microlens array 103. If the microlens array 103 isextended by an absorbing diaphragm 163, it is possible to bring theilluminating laser beam entirely on to the diaphragm 163 to generate anextinction. This thus makes it possible, as previously, to extinguishthe beam at the ends of the trajectory.

Simplified Embodiment with a Spatial Filtering Device Consisting of aPinhole Array.

The embodiment of FIG. 28 is a simplified embodiment with laser light,comprising only one galvanometric mirror, and using a pinhole array asthe spatial filtering system. Owing to its simplicity, in particular interms of synchronization and as regards the number of galvanometricmirrors used, it constitutes a preferred embodiment with laserillumination.

The illuminating beam FX coming from the laser 1500 is coupled by meansof a lens 1501 to an optical fiber 1502 which transports it up to thescanning device. It is then transformed into a parallel beam by thelenses 1503, 1504 and 1505. It is refocused by the lens 1506 on thegalvanometric mirror 1507 which reflects it. It passes through the lens1508 whose object focal plane is on the mirror 1507. It reaches themicrolens array 1509 placed in the image focal plane of the lens 1508.

It is split by this microlens array into a plurality of illuminatingsub-beams FX2, which is then reflected by the mirror 1511 and then thedichroic mirror 1512. It passes through the lens 1 513 made up of twoseparate lenses whose separation can be adjusted in order to adjustprecisely the focal length. It is reflected by the mirror 1514 and thenby the galvanometric mirror 1515, passes through a lens 1516 and then adiaphragm 1517. The lens 1516 has one focal plane on the mirror 1515 andanother focal plane on the diaphragm 1517. The lens 1513 has one focalplane in the focusing plane 1510 of the sub-beams coming from themicrolens array 1509, and another focal plane on the galvanometricmirror 1515. The diaphragm 1517 is mounted in an image plane ofmicroscope, and the beam passing through this diaphragm then reaches thesamples, through the tube lens and the objective, these elements notbeing shown in the drawing.

The light beam FE emitted by fluorescence by the sample in turn reachesthe image plane where the diaphragm 1517 is located, via the objectiveand the tube lens. It is reflected by le galvanometric mirror 1515, bythe mirror 1514, passes through the lens 1513 and the dichroic cube1512, is reflected by the mirror 1518, and passes through the pinholearray 1519.

The beam to be detected FD coming from this pinhole array then passesthrough all the mirrors 1520 represented in FIG. 8 in a view along thedirection V. It is reflected by the mirror 1521, passes through the lens1522 identical to the lens 1513, is reflected by the second face of thegalvanometric mirror 1515, passes through the lens 1523, and is focusedon a sensor 1524 located in an image focal plane of the lens 1523.

The device may be modified as indicated in FIG. 29 to enable observationunder reflected white light. In the device of FIG. 29, the illuminatingbeam FX coming from an incoherent light source 1550, for example a Xenonarc lamp, then passes through a lens 1551 and a diaphragm 1552, and thena lens 1553. The illuminating beam then passes through the lens 1554 andreaches the pinhole array 1555 which splits it into a plurality ofsub-beams and which is placed in a common image focal plane of thelenses 1554 and 1513. The dichroic cube 1512 is replaced by awavelength-neutral beamsplitter cube, for example of the semi-reflectingtype. The device thus modified enables observation under reflectedlight. It constitutes a preferred embodiment for observation underreflected light, for which it is preferable to the device in FIG. 22which cannot totally eliminate stray light.

Whether one uses reflected white light or laser-excited fluorescence,the illuminating points in the object must be sufficiently close to eachother and the array of illuminating points in the object must becorrectly oriented in relation to the direction of movement of thesepoints which is generated by the movement of the galvanometric mirror1515. This allows the use of the single-mirror scanning technique basedon the principle already described in FIG. 19. The galvanometric mirror1507 is designed to allow the movement of the illuminated zone over thedevice splitting the beam into sub-beams, which may be either the lensarray 1509 or the pinhole array 1555, to permit the use of the techniquedescribed in FIG. 23, in which the transparent part of the transparentplate 1600 is now replaced by a diaphragm 1556 or 1529, and in which thezone 1601 of this plate is replaced by the pinhole array 1555 or themicrolens array 1509. The galvanometric mirror 1507 must be synchronizedwith the galvanometric mirror 1515, and the amplitude of its movementmust be appropriate so that the illuminated zone in the object,corresponding to the geometrical image of the illuminated zone in theplane 1510 or on the pinhole array 1555, is fixed.

It is also possible not to use the mirror 1507 or to replace it by afixed mirror. The sample scanning technique is then modified and thediaphragm 1517 is used to delimit the observed zone. FIG. 30 illustratesthe scanning technique that can then be used. Because of the movement ofthe only mirror 1515, the illuminated zone on the sample moves. FIG. 30(a) represents the diaphragm 1517 having an aperture 1611, as well as theilluminated zone 1612 which is made up of an array of illuminatedpinholes moving over the diaphragm. When the mirror is in an endposition of its oscillatory movement, the illuminated zone 1612 is inthe position indicated in FIG. 30 and the plurality of illuminatingsub-beams is entirely stopped by the diaphragm. When the mirror rotates,the illuminated zone 1612 moves and passes through the aperture of thediaphragm as indicated in FIG. 30( b). The illuminated zone then reachesthe other side of the diaphragm, as indicated in FIG. 30( c), and itsmovement speed is then cancelled. As the movement speed is cancelledonly for the end positions of the illuminated zone on the diaphragm, inwhich the illuminating beam is entirely stopped by the diaphragm, theaperture of the diaphragm is scanned by points moving at a roughlyconstant speed. The diaphragm 1517 here constitutes a limitation devicestopping the part of the illuminating beam that passes outside of aselection area constituted by its aperture.

The narrower the illuminated zone 1612, the greater the possibility ofminimizing light losses. Nevertheless, the narrower this zone thegreater must be the density of the illuminating point array, thuslimiting how narrow the zone can be. In general, the solution consistingin using the mirror 1507, although more expensive, is technicallypreferable.

The confocal scanning device can, for example, include all the elementscontained in the zone delimited in the figure by the broken line 1525.In this case, the user must simply connect to the scanner a camera, amicroscope, and a source of lighting. However, the confocal scanningdevice can also be limited to the elements delimited in the figure bythe broken line 1526. In this case, the user must also connect to thedevice lenses 1516 and 1523 supplied separately. The advantage of thissolution is that the connection is made in an afocal zone, thus makingit less sensitive to positioning errors, and users can choose the lensused, enabling them for example to modify the system's magnification.

Means for Modifying the Spatial Filtering Characteristics and/orCharacteristics for Splitting the Illuminating Beam into Sub-beams.

In all the embodiments it is possible to modify the spatial filteringcharacteristics and possibly the characteristics of the beam splittingsystem producing sub-beams. For example, the first spatial filteringsystem can be mounted in a movable manner to be exchangeable manually.However, it is often desirable to have the possibility of modifying thespatial filtering system so that it may be driven, for example, by meansof an electric motor.

For example, when the spatial filtering system is a pinhole array, forexample the array 130 in FIG. 6 or the array 1519 in FIG. 29, it can bemade up of two plates 1700 and 1710 sliding against each other, therelative position of these plates allowing the pinhole size to bechecked. The first plate 1700 is represented in FIG. 31. It includes,for example, the pinholes 1701 to 1705. The second plate 1710 isrepresented in FIG. 32 and includes, for example the pinhole 1711. Whenthe two plates 1700 and 1710 are placed one against the other, and whenthe hole 1701 is brought opposite the hole 1711, the spatial filterformed by the two plates together is identical to that which would beformed by the plate 1710 alone, each of the pinholes of the plate 1710being opposite a hole of the same diameter on the plate 1700. When theplate 1700 is moved in the direction of the arrow indicated in FIG. 32,the hole 1702 can be placed opposite the hole 1711. As the hole 1702 hasa diameter smaller than the hole 1711, the diameter of the hole centeredon 1711, in the assembly formed by the two plates 1700 and 1710, islimited by the hole 1707. The assembly formed by the two plates 1700 and1710 is then a pinhole array, but the diameter of each hole in thisarray is that of the hole 1707. By successively bringing the hole 1711opposite the holes 1703, 1704, 1705, it is possible to give differentvalues to the diameter of each pinhole in the array formed by theassembly of the two plates 1700 and 1710 placed against each other. Eachof these two plates may, for example, be obtained by depositing a metallayer on a sheet of glass, followed by an optical lithography operationto obtain the pinholes. When the two plates are placed one against theother, the metal layers of the two glasses must preferably be in directcontact, with the possibility of using an optical oil layer tofacilitate their relative movement. The relative movement of the twoplates can, for example, be controller by means of a positioner using astepping electric motor.

When the spatial filtering system is an array of microscopic mirrors606, as in FIG. 22, it is not possible to use the preceding method. Thespatial filtering system can then be made up of a plate 1720 representedin FIG. 33, and comprising several microscopic mirror arrays 1721 to1726. The plate 1720 can be mounted on a single-dimension positioningdevice comprising preferably a guide rail, and driven by an electricmotor, and allowing movement in the direction of the arrow indicated inFIG. 33. By moving the plate along the arrow indicated in FIG. 33, it ispossible to successively bring each of the zones 1721 to 1726 on to theilluminating beam path. The microscopic mirror arrays 1721 to 1726 maydiffer from each other as concerns the diameter of the microscopicmirrors and/or the director vectors of the arrays, and hence the arraydensity. In an equivalent manner, it is possible to use a plate 1730,represented in FIG. 34, capable of being moved in two directions, andhence capable of including more microscopic mirror arrays. Themicroscopic mirror arrays can each be made on a glass and superposed ona support plate 1720 or 1730. They can also be made directly on thesupport plate. If the spatial filtering system is a pinhole array, anequivalent device can be used in which the microscopic mirror arrays arereplaced by pinhole arrays. Constant and precise positioning of thepinholes is more difficult to obtain with this system than with thesystem made up of two parallel plates represented in FIGS. 31 and 32. Itis thus preferable to use this technique when the spatial filteringsystem plays the simultaneous role of spatial filter and beam splittingdevice, as in the device 606 of FIG. 22 or the device 105 of FIG. 18.

In the other cases, a filter exchange device requiring moderatemechanical precision can also be designed, but the spatial filteringsystem and the device splitting the illuminating beams into sub-beamsmust preferably be grouped in an exchangeable mobile unit. For example,in the device of FIG. 16, the pinhole arrays 105 and 130 as well as thedichroic mirror 104 can be grouped in a single unit represented in FIG.17. Several of these units can be associated as indicated in FIG. 35which shows a set of three units 1741, 1742, 1743 grouped together andmovable as a unit. FIG. 35( a) is a side view (same observationdirection as FIG. 16) and FIG. 35( b) is a side view along the directionV indicated in FIG. 16. By moving the assembly in the directionindicated in FIG. 35( b), it is possible to bring one or the other ofthe three units on to the light beam path. The assembly may be includedin a mechanical device which provides among other things suitablediaphragming so that the cubes not used do not receive any light, andallowing motor-driven movement. The cubes 1741 to 1743 may differ fromeach other by the diameter of the pinholes or the density of the pinholearrays. Each cube includes an array of pinholes 1751 constituting thefirst spatial filtering operation, which is mounted movably togetherwith the beam splitting device and the dichroic mirror. If a microlensarray is used as in FIG. 6, a similar device can be produced, themicrolens array, the spatial filter and the dichroic mirror beingintegrated in a unit, and several units being integrated in a mobilesystem.

INDUSTRIAL APPLICATION

The present optical scanning device, integrated in a confocalmicroscope, can be used for fluorescence and real-time imaging ofbiological objects. Equipped with laser illumination, it allows imagingat the highest speed. Equipped with incoherent lighting, it allowsfluorescence imaging with a complete range of excitation wavelengths.The confocal image obtained by means of the present device lends itselfwell to the use of three-dimensional deconvolution algorithms enablingimproved resolution. The present optical scanning device, in an adaptedversion, can also be used for reflection imaging, for example for theobservation of circuits in microelectronics, or for in-depth observationof living specimens (skin, for example), in which case it is the lightbeam diffracted back by the observed object which is measured.

1. Confocal optical scanning device for viewing an observed area of anobserved object; comprising means (1509) for splitting an illuminatingbeam (FX) into a plurality of illuminating sub-beams (FX2); comprisingoptical means (1513, 1516) for focusing the illuminating sub-beams on toa plurality of illuminating points in the observed object, forming atwo-dimensional array of illuminating points in the observed object;comprising optical means (1513, 1516) for focusing a plurality ofluminous sub-beams (FE) coming back from the illuminating points on to aplurality of luminous points in a first image plane (P1); comprising aspatial filtering system (1519) arranged in the first image plane andcomprising a plurality of microscopic selection areas, each selectionarea being adapted to select the light reaching a corresponding luminouspoint, thereby obtaining a corresponding sub-beam (FD) to be detected;comprising optical means (1522, 1523) for focusing the plurality ofsub-beams to be detected on to a plurality of points to be detected in asecond image plane; comprising at least one rotatably mounted mobilemirror (1515) reflecting: i) the plurality of illuminating sub-beamsdirected towards the observed object, to allow the plurality ofilluminating points to scan the observed object, ii) the plurality ofluminous sub-beams, to allow the plurality of luminous points to befixed in the first image plane, and iii) the plurality of sub-beams tobe detected, to allow the plurality of points to be detected to scan thesecond image plane; and further comprising means to modify the spatialfiltering system.
 2. Confocal optical scanning device as claimed inclaim 1, further characterized in that the means to modify the spatialfiltering system is adapted to modify the size of the microscopicselection areas.
 3. Confocal optical scanning device as claimed in claim1, the microscopic selection areas being microscopic holes.
 4. Confocaloptical scanning device as claimed in claim 1, the microscopic selectionareas being microscopic mirrors.
 5. Confocal optical scanning device asclaimed in claim 1, the means to modify the spatial filtering systembeing a means to exchange a first spatial filtering system for a secondspatial filtering system.
 6. Confocal optical scanning device as claimedin claim 5, the second spatial filtering system being attached to thefirst spatial filtering system.
 7. Confocal optical scanning device asclaimed in claim 5, the spatial filtering system being attached to themeans for splitting and to a beamsplitter.
 8. Confocal optical scanningdevice as claimed in claim 5, the means for splitting being made up ofthe spatial filtering system, and the illumination beam being anon-coherent beam.
 9. Confocal optical scanning device as claimed inclaim 8, further characterized in that the means to modify the spatialfiltering system is adapted to modify the number of the microscopicselection areas.
 10. Confocal optical scanning device as claimed inclaim 8, the microscopic selection areas being microscopic mirrors. 11.Confocal optical scanning device as claimed in claim 1, the spatialfiltering system being made up of a first absorbing andlor reflectingplate (1700), provided with a plurality of pinholes (1701, 1702), and asecond plate (1710) comprising absorbing and/or reflecting parts, andtransparent parts (1711), wherein these two plates are placed oneagainst the other and mobile in relation to each other, so that theabsorbing and/or reflecting parts of the second plate shut off part ofthe pinholes of the first plate, and so that the relative movement ofthe two plates allows the modification of the pinholes left free. 12.Confocal optical scanning device as claimed in claim 1, wherein themeans for splitting the illuminating beam into illuminating sub-beams isa microlens array, further comprising an auxiliary pinholes array placedin a plane where each of the illuminating sub-beams are focused, reachedby the illuminating sub-beams before they reach the rotatably mountedmobile mirror, and wherein said auxiliary pinholes array is adapted tospatially filter each of the illuminating sub-beams.
 13. In theoperation of a confocal scanning microscope for the observation of anobserved area of an observed object, a method comprising the steps of:a) directing an illuminating beam onto an illuminated zone on asplitting area; b) splitting the illuminating beam having reached thesplitting area into a plurality of illuminating sub-beams; c) reflectingthe plurality of illuminating sub-bean3s onto a rotatably mounted mobilemirror; d) focusing the illuminating sub-beams onto a plurality ofilluminating points in the observed object; e) scanning the plurality ofilluminating points over the observed object; f) reflecting on therotatably mounted mobile mirror a plurality of luminous sub-beams (FE)coming back from the illuminating points; g) focusing the plurality ofluminous sub-beams (FE) on to a plurality of luminous points in a firstimage plane (P1); h) descanning the plurality of luminous points, usingthe rotation of the rotatably mounted mobile mirror; i) selecting thelight reaching a plurality of selection areas, wherein each selectionarea selects light reaching a luminous point, to obtain a plurality ofsub-beams (FD) to be detected; j) reflecting the plurality of sub-beamsto be detected on the rotatably mounted mobile mirror; k) focusing theplurality of sub-beams to be detected on to a plurality of points to bedetected in a second image plane; l) rescanning the plurality of pointsto be detected over the second image plane, using the rotation of therotatably mounted mobile mirror, to obtain a first image in the secondimage plane; m) modifying the selection areas; and n) repeating allsteps to obtain a second image in the second image plane.
 14. The methodof claim 13, wherein said step of modifying said selection areas is astep of modifying the size of each of said selection areas.
 15. Themethod of claim 13, wherein said step of modifying said selection areasis a step of modifying the number of said selection areas.
 16. Confocaloptical scanning device for viewing an observed area of an observedobject; comprising a microlens array (1509) for splitting anilluminating beam (FX) into a plurality of illuminating sub-beams (FX2);comprising a pinholes array arranged in a plane in which theilluminating sub-beams are focused, and arranged to spatially filtereach illuminating sub-beam; comprising optical means (1513, 1516) forfocusing the illuminating sub-beams on to a plurality of illuminatingpoints in the observed object, forming a two-dimensional array ofilluminating points in the observed object; comprising optical means(1513, 1516) for focusing a plurality of luminous sub-beams (FE) comingback from the illuminating points on to a plurality of luminous pointsin a first image plane (P1); comprising a spatial filtering system(1519) arranged in the first image plane and comprising a plurality ofmicroscopic selection areas, each selection area being adapted to selectthe light reaching a corresponding luminous point, thereby obtaining acorresponding sub-beam (FD) to be detected; comprising optical means(1522, 1523) for focusing the plurality of sub-beams to be detected onto a plurality of points to be detected in a second image plane;comprising at least one rotatably mounted mobile mirror (1515)reflecting: i) the plurality of illuminating sub-beams directed towardsthe observed object , to allow the plurality of illuminating points toscan the observed object, ii) the plurality of luminous sub-beams, toallow the plurality of luminous points to be fixed in the first imageplane, and iii) the plurality of sub-beams to be detected, to allow theplurality of points to be detected to scan the second image plane. 17.Confocal optical scanning device for viewing an observed area of anobserved object; comprising means (1509) for splitting an illuminatingbeam (FX) into a plurality of illuminating sub-beams (FX2); comprisingoptical means (1513, 1516) for focusing the illuminating sub-beams on toa plurality of illuminating points in the observed object; comprisingoptical means (1513, 1516) for focusing a plurality of luminoussub-beams (FE) coming back from the illuminating points on to aplurality of luminous points in a first image plane (P1); comprising aspatial filtering system (1519) arranged in the first image plane andcomprising a plurality of microscopic selection areas, each selectionarea being adapted to select the light reaching a corresponding luminouspoint, thereby obtaining a corresponding sub-beam (FD) to be detected;comprising optical means (1522, 1523) for focusing the plurality ofsub-beams to be detected on to a plurality of points to be detected in asecond image plane; comprising at least one rotatably mounted mobilemirror (1515) reflecting: i) the plurality of illuminating sub-beamsdirected towards the observed object, to allow the plurality ofilluminating points to scan the observed object, ii) the plurality ofluminous sub-beams, to allow the plurality of luminous points to befixed in the first image plane, and iii) the plurality of sub-beams tobe detected, to allow the plurality of points to be detected to scan thesecond image plane; and further comprising a beam attenuator andattenuator control means for varying the intensity of the illuminatingbeam during each scan of the illuminating points over the observedobject.
 18. The confocal optical device of claim 17, furthercharacterized in that the attenuator control means and beam attenuatorcontrol the beam attenuation so as to eliminate illumination variationsdue to variations in the scanning speed.
 19. The confocal optical deviceof claim 18, further characterized in that the scanning trajectory of anilluminating point over the observed object is made up of at least onesegment, and in that the ratio Ilas/Vscan in each segment dependslinearly on the position of the illuminating point on said segment,wherein ilas is the intensity of the laser beam and Vscan is thescanning speed of the illuminating beam.
 20. The confocal optical deviceof claim 19, wherein the ration Ilas/Vscan is a constant over the entirescanning trajectory.
 21. The confocal optical device of claim 17,further characterized in that the attenuator control means and beamattenuator control the beam attenuation so as to make the time-averagedillumination substantially constant over an observed area of theobserved object.
 22. In the operation of a confocal scanning microscopefor the observation of an observed area of an observed object, a methodcomprising the steps of: a) directing an illuminating beam onto anilluminated zone on a splitting area; b) splitting the illuminating beamhaving reached the splitting area into a plurality of illuminatingsub-beams; c) reflecting the plurality of illuminating sub-beams onto arotatably mounted mobile mirror; d) focusing the illuminating sub-beamsonto a plurality of illuminating points in the observed object; e)rotating the mobile mirror to scan the plurality of illuminating pointsover the observed object; f) varying the intensity of the illuminatingbeam during the scan of the illuminating points over the observedobject, g) reflecting on the rotatably mounted mobile mirror a pluralityof luminous sub-beams (FE) coming back from the illuminating points; h)focusing the plurality of luminous sub-beams (FE) on to a plurality ofluminous points in a first image plane (P1); i) descanning the pluralityof luminous points, using the rotation of the rotatably mounted mobilemirror; j) selecting the light reaching a plurality of selection areas,wherein each selection area selects light reaching a luminous point, toobtain a plurality of sub-beams (FD) to be detected; k) reflecting theplurality of sub-beams to be detected on the rotatably mounted mobilemirror; l) focusing the plurality of sub-beams to be detected on to aplurality of points to be detected in a second image plane; and m)rescanning the plurality of points to be detected over the second imageplane, using the rotation of the rotatably mounted mobile mirror, toobtain a first image in the second image plane.
 23. The method of claim22, further characterized in that the intensity of the illuminating beamis varied so as to eliminate illumination variations in the observedobject due to variations in the scanning speed.
 24. The method of claim23, further characterized in that the scanning trajectory of anilluminating point over the observed object is made up of at least onesegment, and in that the laser beam intensity ilas is varied so thatratio Ilas/Vscan in each segment depends linearly on the position of theilluminating point on said segment, wherein Vscan is the scanning speedof the illuminating beam.
 25. The confocal optical device of claim 24,wherein the ration Ilas/Vscan is a constant over the entire scanningtrajectory.
 26. The method of claim 22, further characterized in thatthe intensity of the illuminating beam is varied so as to make thetime-averaged illumination substantially, constant over an observed areaof the observed object.